Who We Are
The Penn Equine Assisted Reproduction Laboratory (PEARL) at New Bolton Center is part of the University of Pennsylvania’s School of Veterinary Medicine. The Laboratory is headed by Dr. Hinrichs. Matheus Felix, DVM, is the Laboratory's chief embryologist, and the Laboratory’s program coordinator is Kim Gleason, PhD. PEARL is expanding its personnel to include post-doctoral trainees, research assistants, fellows, technicians, and graduate students.
PEARL was founded in 2020, when Dr. Hinrichs accepted the position of the Harry Werner Endowed Professor of Equine Medicine and Chair of the Department of Clinical Studies-New Bolton Center and relocated, with her laboratory, from her previous institution, Texas A&M University. This is a return home for Dr. Hinrichs, as she completed her residency and graduate training at New Bolton Center before embarking on her academic career.
What We Do
PEARL performs both research and clinical work in equine assisted reproduction. PEARL’s clinical equine ICSI program works in collaboration with clinicians in the Section of Reproduction and Behavior at New Bolton Center, to produce foals from client horses -- for problem mares for which embryo transfer has poor success, for stallions with low sperm reserves, and for embryo production post mortem from mares that suffer untimely death. PEARL also accepts oocytes collected by referring veterinarians and shipped to the Laboratory. Other clinical procedures include embryo biopsy for genetic diagnosis, embryo vitrification, and tissue culture and cell line freezing for genetic preservation.
The Laboratory conducts research into aspects of equine gamete biology, fertilization, and embryo development. This includes studies on sperm capacitation, oocyte recovery and maturation, fertilization in vitro, including ICSI and standard IVF, and equine embryo development.
The overall goal of the Penn Equine Assisted Reproduction Laboratory is to provide answers for important clinical problems in equine reproduction, to allow breeders to optimize their breeding programs and to preserve exceptional equine genetics.
All proceeds from the clinical service at PEARL go to support research on equine assisted reproduction in the Laboratory.
PEARL Faculty
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- Katrin Hinrichs, DVM, PhD, DACT
- Professor of Reproduction
- Harry Werner Endowed Professor of Equine Medicine
- Chair, Department of Clinical Studies-New Bolton Center
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PEARL Staff
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- Matheus Felix, DVM
- PEARL Chief Embryologist
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- Kim Gleason, PhD
- Embryologist & Equine ICSI Program Coordinator
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Intracytoplasmic Sperm Injection Program
ICSI Program Purpose
The intracytoplasmic sperm injection (ICSI) program is offered at the University of Pennsylvania as a means of establishing pregnancies from oocytes (eggs) recovered from mares. Using ICSI, oocytes are injected with individual sperm from a chosen stallion, and the resulting embryos are allowed to develop in the laboratory for approximately one week. Developed embryos are then vitrified (frozen) so that they can be shipped to an embryo transfer facility for transfer to a recipient mare, as for standard embryo transfer.
This advanced reproductive technology is appropriate for mares that are unable to become pregnant themselves (e.g., performance mares, mares with chronic uterine disease, cervical lacerations, or other damage to the reproductive tract that prohibit the mare from conceiving or supporting an embryo in the uterus for any length of time). The procedure can also be performed when pregnancies are sought from limited sperm supplies.
We recommend that the ICSI procedure only be used on mares that are not suitable candidates for routine embryo transfer, or for stallions for which sperm cannot be utilized effectively with standard insemination techniques. Because of the expense of the technology involved and the amount of labor associated with ICSI, foals produced from this program should be valuable enough to justify the increased effort and expense to produce offspring. Before participating in the ICSI program, it is important for each owner/lessee to know the regulations of their breed registry regarding the possibility of registering any resulting foals.
Procedure Overview
ICSI can be performed on oocytes collected from live mares or on oocytes recovered from ovaries post-mortem. For work with ovaries from deceased mares at New Bolton Center, the ovaries are typically received by on-call clinicians in the Section of Reproduction and Behavior, and the ovaries are dissected to recover oocytes. Alternatively, referring veterinarians that are equipped for the procedure can collect oocytes from the ovaries and ship the oocytes to the laboratory.
The oocytes are cultured in a hormone-containing medium, to induce maturation; this mimics the natural events that occur 1-2 days prior to ovulation. Our in-vitro maturation process generally takes 24 to 30 hours, depending upon the quality of the recovered oocytes.
After this culture period, oocytes that have matured are injected with individual sperm from the desired stallion. For this procedure, one sperm is injected into the cytoplasm of each oocyte under a high-power microscope. The resulting fertilized oocytes are cultured in the laboratory to allow development into blastocysts; that is, embryos suitable for transfer to the uterus of a recipient mare. The blastocysts are then vitrified (frozen) for storage or shipment to a center for warming and transfer to recipient mares, or can be shipped fresh if owners have a method for transport.
Ovary Collection and Shipment
For methods for post-mortem ovary collection, handling, and shipment, please visit the Reproductive Service. It is important to know that ovaries should NOT be refrigerated. Leave at room temperature or slightly cooler.
Anticipated Results
Typically, 10 to 15 oocytes are recovered from one pair of ovaries post-mortem; however, this number is variable and is typically lower if the mare is old or has been chronically ill.
From our past results with oocytes recovered from mares post-mortem, on average, 50% of the recovered oocytes should mature in culture, and these will undergo fertilization by ICSI. After fertilization, 20% of fertilized oocytes are expected to develop to the blastocyst stage in the laboratory. After vitrification, warming and transfer, approximately 50 % of the transferred blastocysts are expected to establish ongoing pregnancies. Taken altogether, this gives us about a 50% chance of normal pregnancy for every 10 oocytes recovered. These percentages will decrease if the mare is old, has been ill or debilitated, if the ovaries are not handled at the correct temperature, or if oocytes are delayed in being recovered from the ovaries. Results can also vary depending on the quality of the sperm used for the procedure.
Costs: If you would like to know more about costs for our services, please contact our Program Coordinator, Kim Gleason, PhD, in the lab at 610-925-6575. Or, you can send an email to Kim at kimgl@upenn.edu.
Incidental charges: Any costs for semen collection or shipment of semen containers or other charges not covered by the above information are charged to the client separately.
Cell Line Processing
Obtaining a Tissue Sample for Cell Culture and Freezing
- At collection, the tissue will be placed directly into cold culture medium (DMEM/F-12 with 10% fetal bovine serum and antibiotics). We can send the medium; it is sent chilled in three 15-ml conical tubes. Alternatively, chilled embryo holding medium may be used; using sterile technique, fill three 5- to 15-ml sterile sealable tubes 3⁄4 full with medium.
- The tubes of medium should be refrigerated until use. When going to take the tissue sample, the tubes should be placed in an insulated (e.g. Styrofoam) container (with cover), on ice,.
- Before going to take the sample, make sure you have something to label the tubes with, and that there is someone ready to open the tubes, who has a 1.5 inch 18- or 20-ga needle ready to help get the tissue off the forceps.
- We typically take tissue from the neck under the mane for cosmetic reasons and because this may have had less sun exposure. For an animal that may be terminal, it is recommended that two different areas be sampled, and the tissue grown as two separate cell lines. In this case take another sample from the gum if possible, or if not, from any other place easy to get to, which has been out of the sun. Try to avoid really fatty areas such as the tail head.
- Shave an area approximately 7 x 7 cm (unless gum), and scrub and rinse as for surgery. We typically use betadine scrub rather than nolvasan. Do a last wipe with only sterile saline to remove remnants of scrub. For gum, wipe with betadine and then with sterile saline.
- Glove and maintain sterility; contamination is the most common problem with tissue biopsies.
- Perform an inverted U block with lidocaine, so that the lidocaine is not in the tissue being sampled.
- Make a small skin incision (about 2 cm), and evert the skin edges. Obtain small (~5 mm3) samples of subcutaneous connective tissue and place them immediately into the cold cell culture medium. Place 1 or 2 pieces of tissue in each tube. The assistant, using the needle, should help move the tissue quickly, and should make sure the tissue goes down into the medium. If you think any contamination may have taken place, start with a new tube, and new instruments if necessary.
- If taking samples from two areas, put the samples in different tubes and label accordingly.
- Keep the medium and samples cold, and cover the container so the tubes are not exposed to sun or fluorescent light. Transport as quickly as possible to the laboratory. Shipping address:
Kim Gleason, Program Coordinator
Penn Equine Assisted Reproduction Laboratory
Department of Clinical Studies – New Bolton Center
University of Pennsylvania School of Veterinary Medicine
382 West Street Road
Kennett Square, PA 19348
Phone: 610-925-6575
Email: kimgl@upenn.edu
- Close incision with a couple of sutures.
Equine Embryo Vitrification
Purpose of Equine Embryo Vitrification
Embryo vitrification offers a means to cryopreserve (freeze) embryos from donor mares. Embryo vitrification has long been successful in small embryos, such as those flushed from mares on Day 6 after ovulation. We have developed a method to cryopreserve larger, Day-7 or Day-8 embryos (blastocysts). Using our vitrification method, blastocysts larger than 300 μm in diameter, which are fluid-filled, are collapsed by puncturing their outer layer. This manipulation allows the embryo to be vitrified successfully. The vitrified embryos are placed in liquid nitrogen and can be stored indefinitely. Collapsed, vitrified embryos reform their shape quickly when warmed, and have resulted in good pregnancy rates (70%) after transfer.
This advanced reproductive technology is useful in equine industries that desire foaling dates early in the year, and also has the advantage of using embryos recovered from donor mares at the standard time after ovulation (Day 7 or 8). Embryo vitrification makes it possible to attain earlier foals from mares that, due to age or chronic breeding issues, do not provide embryos until late in breeding season, or from mares that foal late in the breeding season. The vitrified embryos can be warmed and transferred in the following years, at a time of the embryo owner's choice.
Please Note:
- We do not store embryos here in our laboratory; once they are vitrified, they are shipped to you or to an embryo storage facility that will bill you directly for charges related to storage.
- Before participating in the embryo vitrification program, it is important for each owner/lessee to know the regulations of their breed registry regarding the possibility of registering any resulting foals, and any effect of transferring an embryo in a year other than that of the recorded breeding.
The amount billed by PEARL only covers the charges for vitrification, blastocoele collapse, genetic biopsy (see below), warming and shipping. All charges related to the transfer of vitrified/warmed embryos to recipient mares will be billed to you, the client, by the embryo transfer facility performing the transfer and are not included.
Biopsy for Genetic Diagnosis
Our laboratory also offers the option for embryo biopsy, which is, taking a small sample of cells from the embryo for genetic analysis, before vitrification. For more information, see Embryo Biopsy for Genetic Diagnosis
Costs: If you would like to know more about costs for our services, please contact our Program Coordinator, Kim Gleason, PhD, in the lab at 610-925-6575. Or, you can send an email to Kim at kimgl@upenn.edu.
Embryo Biopsy for Genetic Diagnosis
PEARL offers our clients the ability to take a genetic sample from an embryo before it is transferred. Embryos may be shipped to the Laboratory for biopsy, after being collected from mares at the standard time (Day 7 after ovulation).
Embryo biopsy offers the owner the option of knowing the genetic makeup of an embryo before it is transferred. By knowing this, the owner can avoid production of a foal affected with a genetic disease, or even avoid transferring an embryo carrying the gene for a disease. In addition, the genetic analysis can determine the sex, coat color, and parentage verification of the upcoming foal.
The biopsy is performed by taking a small sample of cells from the embryo using a micromanipulator and micropipette. The cells are collected from the area of the embryo that eventually forms the placenta. Approximately 20 cells are collected; because the typical embryo at Day 7 contains thousands of cells, there is minimal effect on the embryo. The collected cells are then submitted to a reference laboratory for genetic analysis. After the cells have been collected from an embryo, the embryo is typically vitrified (frozen) to be transferred after the genetic analysis results are available (about one week).
The genetic analysis is >95% accurate; however, there is a slight possibility (<5%) of a gene being missed or the DNA not being read.
Embryos that have been subjected to biopsy then transferred have a normal pregnancy rate (about 80% in our studies). There is no increase in pregnancy loss after transfer of biopsied embryos, and foals born from biopsied embryos are normal.
Goszczynski DE, Tinetti PS, Choi YH, Hinrichs K and Ross PJ. Genome activation in equine in vitro-produced embryos. Biol. Reprod. 106: 66–82 (2022).
Goszczynski DE, Tinetti PS, Choi YH, Ross PJ and Hinrichs K. Allele-specific expression analysis reveals conserved and unique features of preimplantation development in equine ICSI embryos. Biol. Reprod. 105:1416–142 (2021). Featured as BOR Editor’s Choice for December 2021 issue.
Sampaio B, Ortiz I, Resende H, Felix M, Varner DD and Hinrichs K. Factors affecting intracellular calcium influx in response to calcium ionophore A23187 in equine sperm. Andrology 9:1631-1651 (2021).
Brom-de-Luna JG, Salgado RM, Felix MR, Canesin HS, Stefanovski D, Diaw M and Hinrichs K. Culture protocols for horse embryos after ICSI: effect of myo-inositol and time of media change. Anim. Reprod. Sci. 233:106819 (2021).
Ortiz I, Felix M, Resende HL, Ramirez-Agámez L, Love CC and Hinrichs K. Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the “zombie sperm” dilemma. J. Assist. Reprod. Genet. 38:2465-2480 (2021).
Lewis N, Hinrichs K, Leese HJ, McG Argo C, Brison DR and Sturmey R. Glucose concentration during equine in vitro maturation impacts mitochondrial function. Reproduction 160:227-237 (2020).
Canesin HS, Ortiz I, Nascimento Rocha Filho A, Salgado RM, Brom-de-Luna JG and Hinrichs K. Effect of warming method on embryo quality in a simplified equine embryo vitrification system. Theriogenology 151:151-158 (2020).
Lewis N, Hinrichs K, Leese HJ, McG Argo C, Brison DR and Sturmey R. Energy metabolism of the equine cumulus oocyte complex during in vitro maturation. Sci. Rep. 10:3493, doi.org/10.1038/s41598-020-60624-z (2020).
Angel D, Canesin HS, Brom-de-Luna JG, Morado S, Dalvit G, Gomez D, Posada N, Pascottinie OB, Urregoa R, Hinrichs K and Velez IC. Embryo development after vitrification of immature and in vitro-matured equine oocytes. Cryobiology 92:251-254 (2020).