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.

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted, frozen–thawed spermatozoa in field conditions

“…However, despite the feasibility of the sex‐sorting procedure having been described by several authors (Meyers et al . ; Rodenas et al . ), the technique for AI and the high‐dose insemination of the number of sex‐sorted sperm preclude the practical application of this procedure at the farm level.…”

“…Different traits and temperaments that are sex‐related often underlie the selection of dogs used for various applications by breeders and owners with different demands (Meyers et al . ).…”

“…Similar applications in the canine industry have yet to occur, as only a few puppies have been produced from fresh (unfrozen) sorted spermatozoa (Meyers et al . ). To date, the successful cryopreservation of sorted spermatozoa and subsequent production of offspring after artificial insemination (AI) has not been reported in canines.…”

“…For dogs, interest in predetermining the sex has been generated from those with pets and has applications for working dogs, such as guide dogs, military and police dogs, guard dogs and tracking dogs (Garner & Seidel 2003;Oi et al 2013). Different traits and temperaments that are sex-related often underlie the selection of dogs used for various applications by breeders and owners with different demands (Meyers et al 2008).…”

Artificial Insemination in Veterinary Science

“…Since then, DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species (Garner, 2006;Garner et al, 1983;Lu et al, 2010;Pinkel et al, 1982), and offspring have been produced from sexed sperm of at least seven species, including rabbits (Johnson et al, 1989), humans (Levinson et al, 1995), cattle (Cran et al, 1993), horses (Buchanan et al, 2000), sheep (Catt et al, 1996), dogs (Meyers et al, 2008), cats , elk (Schenk & DeGrofft, 2003), buffalo (Presicce et al, 2005) and dolphins (O'Brien & Robeck, 2006). The first offspring born with flow cytometrically sex sorted spermatozoa was in rabbits after surgical AI into the oviduct (Johnson et al, 1989).…”