266 BIRTH OF CANINE OFFSPRING FOLLOWING INSEMINATION OF A BITCH WITH FLOW-SORTED SPERMATOZOA

Abstract

Different traits and temperaments that are sex-related often underlie the selection of guide dogs used for auditory and/or visually impaired humans. The objective of this project was to select X-chromosome-bearing canine sperm by means of flow cytometry/cell sorting based on DNA content (X chromosome- bearing canine sperm contain 3.7% more DNA than Y-chromosome-bearing sperm) and use the sorted sperm for artificial insemination (AI) to produce live offspring. This technology has proven reliable for sex selecting offspring using AI in a number of species and is of great commercial value to food animal livestock production. To test the efficacy of sorted canine sperm, a 2-year-old nulliparous Labrador retriever bitch in observed heat was monitored for plasma progesterone levels for a baseline rise to 2.8 ng mL^–1, indicative of the LH surge. Progesterone levels more than doubled (5.8 ng mL^–1) 2 days later, suggesting ovulation. One ejaculate was collected from a proven Labrador retriever stud on each of Days 2, 5, and 6 following the estimated LH peak. Sperm (200 million) were diluted in TRIS buffer without egg yolk (pH 7.4) and stained with 97.4 µm Hoechst 33342 in 1-mL aliquots for 45 min in a 34.5°C water bath and then diluted to 100 million sperm mL^–1 with 4% egg yolk TRIS (pH 6.2) containing 0.002% food coloring dye (FD&C #40). Sperm were sex-sorted using an SX MoFlo™ sperm sorter (Dako, Fort Collins, CO, USA), operated at 40 psi and using a TRIS-based medium (pH = 6.8, 370 mOsm kg^–1 water) as sheath fluid: 10 000–20 000 events s^–1 and sort rates of 800–2000 sperm s^–1. Sorted sperm were concentrated by centrifugation for 20 min at 850g and, after the supernatant was removed, were extended with 20% egg yolk TRIS extender (pH 6.8) to a total volume of 2 mL. Transcervical insemination of fresh, sorted sperm occurred within 9 h of the semen collection. An additional 1 mL of 20% egg yolk TRIS extender was used to flush the insemination catheter. The respective number of X-chromosome-bearing sperm inseminated per day was 46.2, 20.3, and 18.2 million, totaling 84.7 million sperm, and the weighted purity mean across the 3 sort days was 82%. Pregnancy was ascertained by ultrasound 30 days post-AI. Five puppies (3 female and 2 male) were delivered via natural birth 62 days after first AI. Only 60% of the offspring were of the predicted sex due to an 18% probability of chance, given the weighted mean purity of the sorted sperm and for a litter of 5 puppies. This demonstrates that fresh canine sperm can be separated by sex on the basis of DNA content, and transcervical insemination of low numbers of non-frozen sorted canine sperm can be used to produce live offspring.

This work was funded by XY, Inc.