Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
IMPACT OF ABNORMAL SPERM ON EARLY EMBRYO DEVELOPMENT FOLLOWING IVF AND ICSI
 
PROJECT DIRECTOR: Gwazdauskas, F. C.
 
PERFORMING ORGANIZATION
DAIRY SCIENCE
VIRGINIA POLYTECHNIC INSTITUTE
BLACKSBURG,VA 24061
 
NON TECHNICAL SUMMARY: Semen characteristics are vital to providing gametes that are capable of fertilization. We wish to refine techniques that will allow for greater reproductive efficiency in cattle. With increases in reproductive efficiency by reducing days open through better predictive measures of semen evaluation, we estimate that a reduction by 3 days can save dairy farmers $1.08 million annually in the state.
 
OBJECTIVES: The long term goals of our research are directed toward increasing reproductive performance in cattle. We aspire to develop and refine techniques and technologies that will allow for increased reproductive productivity at the stakeholder level and have application to emerging biotechnologies. In this regard, this proposal addresses primarily fundamental-linked research on gamete physiology, fertilization, in vitro embryonic survival, and enhancement of systems involved with emerging reproductive biotechnologies. Therefore, the objective of this study is to implement assisted reproductive technology to determine the effect of fertilization with a sperm cell population containing a high number of morphological abnormal sperm cells on embryonic development. In order to address the overall objective, experiments will be conducted implementing in vitro fertilization (IVF) procedures with morphological abnormal spermatozoa to eliminate the selection of the female tract. Additionally, we will implement intracytoplasmic sperm injection (ICSI) procedures with spermatozoa having specific abnormalities to determine the ability of these spermatozoa to undergo pronuclear formation and sustained subsequent embryo development when requirements to initiate fertilization are eliminated. Individual experiments will focus on addressing the following questions (the first question relates to IVF experiments only). 1) Is there a difference in the ability among presumptive zygotes to undergo cleavage and subsequent cell division following fertilization with normal and abnormal spermatozoa? 2) Does subsequent embryo development differ between IVF with an abnormal sperm population and after injection of a single sperm with a specific abnormality into an oocyte? 3) Are there differences in embryonic development related to natural occurring cell death (apoptosis) that might be more readily observed by using morphological abnormal spermatozoa?
 
APPROACH: Experiments will be conducted implementing IVF procedures with morphological abnormal spermatozoa. This phase will be followed by experiments implementing ICSI procedures with spermatozoa having specific abnormalities. In vitro fertilization will be performed with frozen-thawed semen samples from Holstein bulls. Morphological abnormalities present in these samples were induced through a 48 h scrotal insulation. Samples collected prior to scrotal insulation (d -8, -5 and -1) will serve as control samples. Samples were collected every other day after insulation for a period of 34 d. Experiment I. Oocytes (n =200/trip) obtained from an abattoir will be randomly assigned to different groups (n = 4) that will serve as the various treatments (Trt): Trt 1: IVF with a sample from a non-treated bull (n = 50 oocytes); Trt 2: IVF with a pre-insult sample from bull A (d -8, -5, or -1; n = 50); Trt 3: IVF with the perturbed sample bull A collected on d 20 (n = 50); and Trt 4: IVF with a perturbed sample bull A collected on d 23 (n = 50). Six replicates will be performed. Experiment II & III. These will be repetitions of Experiment I using samples from Bulls B and C that have different types of defects. Experiment IV-VI. Intracytoplasmic sperm injection will be performed with a single spermatozoon from a population of abnormal spermatozoa from bulls A, B, and C following the design above. Methods. Two to 8 mm follicles will be aspirated from slaughterhouse ovaries. Maturation will be performed in tissue culture medium supplemented with FSH, LH, 17 beta-estradiol, and fetal calf serum (FCS) in a humidified atmosphere of 5% CO2 in air for 22 to 24 hr. For IVF, frozen semen from a pre-selected bull will be used. Semen will be thawed in a water bath. Motile spermatozoa will be separated by a modified swim-up technique. Following maturation the cumulus-oocyte complexes (COC) will be transferred to IVF medium supplemented with 10 ug/ml heparin and BSA and 2 ul of the sperm cells suspension will be added to the IVF medium. After 18 hr of the COC and sperm cell incubation cumulus cells will be removed by vortexing. Presumptive zygotes will be cultured in SOF culture medium supplemented with BSA and FCS will be added after 96 hr of culture. Presumptive zygotes/embryos will be removed from each treatment group at 2-cell, 8-cell and blastocyst stages of development. Embryos will be removed, fixed, transferred to a slide and saved until the appropriate assays (TUNEL and caspase) are performed. Apoptotic Activity. Embryos will be removed from culture medium and washed. Embryos will be incubated in PhiPHiLux-G1D2. Caspase activity will be determined with epifluorescence microscope. Embryos will be removed from culture media at appropriate times for TUNEL and will be fixed in a paraformaldehyde solution, washed, and transferred to poly-l-lysine coated slides and incubated with DNase. In Situ Cell Death Detection Kit fluroescein will be added. Slides will be washed and incubate with RNase A followed by blottting and adding propidium iodide. Confocal microscopy will be used to determine the number of red (total) and green/yellow (apoptotic) cells.
 
CRIS NUMBER: 0197687 SUBFILE: CRIS
PROJECT NUMBER: VA-135707 SPONSOR AGENCY: CSREES
PROJECT TYPE: HATCH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Oct 1, 2003 TERMINATION DATE: Sep 30, 2008

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
301331010202.210%
301331010302.210%
301331010402.210%
301331010502.210%
301341010202.210%
301341010302.210%
301341010402.210%
301341010502.210%
301351010302.210%
301351010502.210%

CLASSIFICATION HEADINGS
KA301 - Reproductive Performance of Animals
S3310 - Beef cattle, live animal
S3510 - Swine, live animal
S3410 - Dairy cattle, live animal
F1020 - Physiology
F1030 - Cellular biology
F1050 - Developmental biology
F1040 - Molecular biology
G2.2 - Increase Efficiency of Production and Marketing Systems


RESEARCH EFFORT CATEGORIES
BASIC 50%
APPLIED 25%
DEVELOPMENTAL 25%

KEYWORDS: in vitro; abnormalities; sperm; embryos; embryo development; fertilization; beef cattle; dairy cattle; reproductive performance; swine; animal physiology; cell biology; molecular biology; embryo survival; biotechnology; new technology; spermatozoa; zygotes; cleavage; apoptosis; cell morphology; semen; reproductive efficiency

PROGRESS: Oct 1, 2006 TO Sep 30, 2007
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.

IMPACT: 2006-10-01 TO 2007-09-30 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.

PUBLICATION INFORMATION: 2006-10-01 TO 2007-09-30
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.

PROJECT CONTACT INFORMATION
NAME: Gwazdauskas, F. C.
PHONE: 540-231-4756
FAX: 540-231-5014