Embryo Recovery Procedures and Collection Success: Results of 492 Embryo-Flush Attempts

1. Introduction

Embryo transfer is a common reproductive technique in clinical veterinary practice. Advantages of embryo transfer are that valuable mares may have more than one foal per year, older mares can donate embryos to young recipients, subfertile mares can donate embryos to reproductively healthy mares, and mares in athletic competition can donate embryos while remaining in training. The first foal produced by embryo transfer was born in 1974. Over the subsequent 36 yr, specialized equipment has been developed and practical techniques were adopted to enhance embryo-collection success. The goal of this paper is to review embryo-recovery techniques and provide data on embryo-collection success in a clinical embryo-transfer program.

Embryo recovery is usually attempted 7 or 8 days postovulation.^1,2  Collection of small (i.e., <300 μm) embryos for cryopreservation necessitates flushing at day 6.5 or early on day 7 after ovulation. A sterile silicone catheter, typically of 8.0-mm internal diameter with an inflatable cuff, is used to facilitate transcervical lavage. A 75-μm embryo filter is either attached to the outflow line or the outflow line is manually regulated at the level of the filter. A sterile Y-tubing set, with clamps to regulate inflow and outflow, is used to connect the catheter, fluid bag, and embryo filter. 

The media used for embryo collection can be a commercially prepared complete flush media that contains a buffer system, antibiotic, and surfactant, or it can be as simple as lactated Ringer’s solution with the optional addition of either calf serum or purified bovine serum albumen (BSA) as a surfactant to prevent adherence of the embryo to the tubing, filter, or search dish.

The uterus of the donor mare is usually lavaged three to four times in sequence using approximately 1 l of fluid each time. The media may be pre- warmed (i.e., 30–35°C) or used at room temperature. The amount of fluid used for each flush is dependent on the size of the uterus and/or parity of the mare. The goal is to expand the uterine lumen enough to allow fluid to effectively reach all parts of the uterus, including the area between or under the endometrial folds. The flush medium is allowed to flow back out of the catheter by gravity flow through the embryo filter. The uterus of the mare may be massaged per rectum during the infusion and recovery of the media. Recovery of the uterine lavage fluid may be monitored by collecting the effluent in a graduated cylinder and/or ultrasonographic examination of the uterus at the end of the procedure.

The search for an embryo may begin after each successive lavage or after the final volume of media is recovered. Contents of the filter are poured into a search dish and examined for the presence of an embryo. If an embryo is not recovered, additional media may be infused into the uterus, and the mare is administered 20 units of oxytocin intravenously to stimulate uterine contractions. The media is allowed to stay in the mare for approximately 3 min before being allowed to exit by gravity flow aided by uterine massage per rectum.^3,4 At the conclusion of the embryo-flush procedure, the donor mare is administered prostaglandins to lyse the corpus luteum.

Embryo-recovery rate is influenced by many factors, such as age and fertility of the donor mare, quality of the sire’s semen, day of recovery, number of ovulations, and clinical expertise.^1,2 Embryo-recovery rate is correlated with age and reproductive status of the donor mare. A higher percentage of embryos are recovered from mares <10 yr of age than from mares >15 yr of age. Embryo-recovery rates seem to be ~5–10% below expected pregnancy rates per cycle.

2. Materials and Methods

A retrospective analysis of embryo-recovery success in the clinical embryo-transfer program at Colorado State University was performed. A total of 492 embryo-recovery procedures performed on client mares at Colorado State University between 2004 and 2008 were reviewed. Data were included only if the reproductive management of the donor mare was performed on site. A total of 257 embryos were collected; in a limited number of cases, embryo-quality score or developmental stage were not recorded. Accurate age of the embryo relative to collection day was not known in cases of asynchronous double ovulations in which a single embryo was collected. Embryos were evaluated for quality, size, and stage of development as previously described.⁵ Comparisons of recovery rates and embryo size between groups were made using statistical analysis software (SAS).a All values are presented as the mean ± SEM.  [...]