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28 Fluorescent Microspheres Test for Evaluation of Oviductal Patency

Sofie Sitters1 and John J. Dascanio2

1 Amsterday, The Netherlands

2 School of Veterinary Medicine, Texas Tech University, USA

Introduction

Blockage of one or both oviducts with intraluminal gelatinous masses can result in reduction of fertility in the mare. The starch granule test has limitations for evaluation of oviductal patency in the mare. One limitation is the inability to evaluate both oviducts simultaneously. A second concern is the size variation of the starch granules that typically seem to pass through the tested oviducts and that these granules are significantly smaller than the diameter of an early equine embryo.

Consequently, fluorescent microspheres have been used as an alternative to evaluate oviductal patency. The clear advantage of using fluorescent microspheres over the starch granule test is that use of two colors of beads can discriminate between patency of the left and right oviduct, making it possible to demonstrate patency of both the mare’s oviducts in a single procedure. A solution containing fluorescent microspheres is deposited onto the surface of the ovary using a transvaginal ultrasound probe and needle or applied into the proximal oviductal lumen via a laparoscope. This technique requires access to either a fluorescent microscope or a flow cytometer.

Equipment and Supplies

Examination gloves, tail wrap, tail rope, non‐irritant soap, bucket, disposable bucket liner, roll cotton, obstetrical sleeves, obstetrical lubricant, twitch, sedation, fluorescent microspheres (15 μm in diameter) in two colors (i.e., red and green). Option 1: ultrasound with transvaginal probe and 60 cm (24 inch) 18 gauge single channel needle. Option 2: laparoscope and 8 Fr catheter. Uterine lavage catheter, 0.9% sterile saline or lactated Ringer's solution, flow cytometer or fluorescent microscope.

Transvaginal Ultrasound Technique

The mare should be in diestrus for the procedure. Estrual mares may have a delay in oviductal transport.

A tail wrap and tail rope are placed and the perineum washed in standard fashion (see Chapter 3 & Chapter 4).

The vaginal vault is rinsed with 500 ml solution of sterile saline containing 0.05% povidone‐iodine followed by a rinse with 450 ml sterile saline containing 50 ml of 2% lidocaine.

A sterilized transvaginal probe is passed into the anterior vagina.

One ovary is grasped per rectum and stabilized near the probe head.

The 18 gauge 60 cm (24 inch) single channel needle is passed along the channel of the probe and the vaginal wall is punctured. A twitch may be applied prior to this or sedation provided to the mare, since puncture of the vaginal wall may cause a response by the mare.

5 ml of sterile saline containing 2 million fluorescent microspheres (Triton Technology, Inc., San Diego, CA, USA) is deposited over the surface of the ovary. One color of fluorescent microspheres may be applied to the left ovary and a different color microsphere is applied to the right ovary.

The uterus is lavaged 24 and/or 48 hours later with 1 liter of sterile saline.

The recovered fluid is centrifuged at 1,500 rpm for 10 minutes.

The pellet is resuspended in 5 ml sterile saline.

The sample is evaluated by flow cytometry for the presence and fluorescence pattern of microspheres. Alternatively, the presence of beads may be detected by fluorescent microscopy.

Laparoscope Technique

Mares are evaluated during diestrus.

The standard preparation for standing flank laparoscopy is performed (see Chapter 24).

The ostium of the oviduct is grasped with forceps.

An 8 Fr polypropylene catheter passed through the biopsy channel is used to cannulate the proximal oviduct.

Approximately 1 ml of solution containing 2 million fluorescent microspheres in 0.5% carboxymethylcellulose is infused into the oviduct (when possible) or spread onto the mucosal surface of the infundibulum.

A second procedure is performed on the opposite ovary using a new catheter and different colored microspheres.

The uterus is lavaged 48 hours after surgery with three 1‐liter flushes of lactated Ringer's solution. The fluid is recovered into a 5‐liter graduated cylinder.

The beads are allowed to settle for 1 hour and the supernatant decanted.

100 ml of fluid is aspirated from the bottom and transferred into two 50 ml centrifuge tubes.

The tubes are centrifuged for 10 minutes at 500× g.

The supernatant is removed, and the pellets are combined and examined for the presence of beads by fluorescent microscopy.

Additional Comments

In a study using laparoscopy, fluorescent beads were recovered from the uterus of 86% of mares with oviducts without plugs (determined by flushing excised oviducts at necropsy) as compared with only 29% of mares with oviducts containing plugs. Interestingly, 92.8% of oviducts contained beads of both colors, suggesting intraperitoneal bead movement. Ultimately, the study noted a 71.4% ability of the test to correctly detect a mare with plugs (test sensitivity) and an 85.7% ability of the procedure to correctly detect a mare without plugs (test specificity). The absence of fluorescent beads in the uterine lumen is suggestive of, but not conclusive evidence for, a blocked oviduct.

As with the starch granule test, the diameter of the microspheres used in the published studies are considerably smaller, at 15 μm, than an oocyte or early developing oviductal stage embryo (150–250 μm) and may not be the ideal size for the detection of a mass that would impede embryo transport distally through the isthmus and into the uterus.

Transvaginal deposition may result in a variable amount of beads entering the infundibulum; whereas, laparoscopic cannulation results in more accurate placement of beads. Laparoscopic deposition does, however, require a certain technical skill and specialized equipment. Laparoscopic deposition of beads also allows for direct observation of the oviduct. Mares with oviductal pathology may exhibit oviductal swelling, and an increase in vascularity or adhesions.