• COS and COv Protocol Management
  • The ART Core staff can oversee the treatment of investigator animals to ensure injection schedules are maintained and animals are properly managed as well as monitor ovarian activity with routine ultrasound imaging.
  • COS: Controlled Ovarian Stimulation is used to produce multiple follicles during a single ovarian cycle
  • COv: Controlled Ovulation allows for the managed development of a single, naturally selected follicle
• Follicle Aspiration and Oocyte Collection
• Following either a COv cycle or a stimulated COS cycle, ART Core personnel can track the animal’s ovarian status through circulating steroid (estradiol and progesterone) levels and ultrasound image analysis, coordinate the surgical collection and provide collection medium, isolate oocytes from the follicle aspirates, and carry out in vitro culture of recovered gametes.Vaginal Swabbing to Confirm Mating
• During timed mated breeding, vaginal swabs may be analyzed to determine success during natural breeding attempts.

• In Vitro Fertilization (IVF)
  • A process whereby semen from investigator animals or ART Core males is incubated in optimized medium with mature oocytes to produce pre-implantation stage embryos.  Typically used to evaluate changes in oocyte quality due to treatment effects associated with contraception or infertility-based research projects.
• Intracytoplasmic Sperm Injection (ICSI)
  • A preferred method for fertilization when the endpoint is embryo production and live offspring. ICSI injects a single spermatozoon into a mature ova using micromanipulation technology.

• The ART Core can perform several procedures to aid in the study of early embryonic development.
• Microinjection
  • This includes injection of oocytes and zygotes with reagents for experimental purposes such as siRNAs, morpholinos, CRISPR/Cas9 or TALENs for gene knockdown and reagents for genome editing.
• Embryo Biopsy
  • The Core can also perform trophectoderm and polar body biopsies to obtain material for genetic analysis.
• Assisted Hatching
  • Trained ART Core personnel can artificially “hatch” pre-implantation blastocyst stage embryos

• Embryo culture
  • Fertilized oocytes can be cultured in vitro up to blastocyst stage, when they can be biopsied, cryopreserved and/or transferred into a recipient.
• Embryo Cryopreservation
  • In vitro produced embryos can be cryopreserved for storage for later use or analysis.
• Embryo Transfer
  • The Core can surgically transfer embryos into appropriately staged recipients.
• Embryo micromanipulation
  • Please refer to “Oocyte/Embryo Micromanipulation” to find out more.

• Semen Collection and Processing
  • Freshly collected semen can be used for artificial insemination when the exact timing of insemination is required. Further processing of the semen sample can isolate and determine the number of motile spermatozoa for in vitro fertilization protocols.
• Advanced Sperm Analysis
  • Sperm samples can be assessed using different methods to determine overall sperm quality. The methods include:
  • Computer Assisted Sperm Analysis (CASA), which generates objective data on sperm motility characteristics, as well as sorts sperm subpopulations, according to user-defined criteria;
  • Live/Dead sperm using molecular probes – this assay evaluates if the plasma membrane is intact or non-intact. Sperm with a non-intact plasma membrane cannot control what comes in and out of the cell and eventually will become immotile and have increased DNA fragmentation;
  • Acrosome/plasma membrane integrity using eosin-fast green stain – this assay evaluates both acrosome integrity and plasma membrane integrity. Sperm with a non-intact plasma membrane cannot control what comes in and out of the cell and eventually will become immotile and have increased DNA fragmentation. Sperm with a non-intact acrosome are unable to fertilize oocytes;
  • Mitochondrial activity using Mitotracker – this assay evaluates mitochondrial membrane potential. The mitochondria are responsible for generating the energy necessary for sperm motility through cell respiration. Sperm with inactive mitochondria are presumably dead;
  • Sperm DNA fragmentation using Halosperm G2 – this assay evaluates DNA fragmentation. Some fragmentation can be corrected by the oocyte after fertilization, but depending on the degree and type of fragmentation, the oocyte cannot correct it and the embryo will be unable to develop or may develop as a defective embryo.
  • Sperm morphology using formol-saline fixed spermatozoa – this assay is performed by analyzing fixed sperm in wet preparations under phase contrast microscopy. Sperm defects have various impacts on the ability of sperm to fertilize the oocytes, depending on where the defect is located – head, midpiece or tail – and the degree of the defect.
• Semen Cryopreservation
  • Semen from genetically valuable males can be collected and stored in the ART Core facility.
• Sperm Chromatin Structure Assay (SCSA)
  • Using flow cytometry, a DNA fragmentation index (DFI) can be measured to determine the degree of DNA damage in a sperm sample.  This assay is useful when investigating potential fertility issues with an individual animal or when assessing damage due to environmental toxicants.

• Media Preparation
  • All media associated with ART Core protocols are available to researchers and modifications can be made at the investigator's request.
• Experimental Design Assistance
  • Members of the ART Core team are always happy to answer questions about the resources available to researchers or work together to develop new technology to accomplish research goals.
• Fetal Sexing
  • In collaboration with the Hennebold lab, fetal sex may be determined from 2 ml of maternal blood as early as day 20 of gestation.
• Artificial Insemination
  • Non-surgical artificial insemination can be performed on investigator animals when the exact timing of insemination is required and timed-mated breeding isn’t an option.
• Pregnancy Confirmation by ultrasound
  • Measurements including crown-rump-length (CRL) and heart rate can be assessed to determine gestational age and fetal health status.
• Protocol Customization
  • New techniques and protocols can be developed to meet specific research needs.

ONPRC ART Core External Rate Sheet September 2024