86 Creating homozygous offspring using oviductal sperm deposition with poor quality cryopreserved semen from a transgenic founder goat

At LFB USA, transgenic founder goats are generated to produce recombinant therapeutic proteins in their milk utilising the company’s rPRO™ Technology. Herd propagation of a founder line is paramount to large-scale therapeutic protein production, and the use of homozygous male(s) improves the efficiency of the breeding scheme. This case report discusses the use of oviducal sperm deposition (OSD) with poor quality sperm while using superovulation of desired donors and oocyte transfer to produce homozygous offspring. The transgenic hemizygous male founder (F₀), determined to have multiple integration sites and mosaicism, was outbred to wild-type females once sexual maturity was reached. Eight hemizygous transgenic females, demonstrating suitable and reproducible expression levels of the recombinant protein, were produced. Subsequently, semen was collected multiple times from the same founder male and cryopreserved, with a post-thaw analysis exhibiting very low motility with only 10 to 20% live sperm. The prospect of using OSD methods for producing homozygous males for large-scale propagation of the expressing line was hypothesised. These same eight hemizygous females were superovulated and their oviducts were surgically flushed to collect a total of 133 unfertilised ova from 220 ovulation points. The low-quality cryopreserved semen from the founder male was then washed and deposited directly into the oviducts of the eight superovulated females. A total of 15 of the 133 ova were then immediately transferred back to the eight hemizygous females. The remaining 118 ova were transferred on the same day to an additional 24 synchronised wild-type recipients after OSD was performed on the oviduct ipsilateral to the ovary without an ovulation point to decrease the fertilisation of the recipients’ ova. Pregnancies were produced in three of the eight hemizygous females and 14 of 24 wild-type recipients. This work generated 26 offspring, with 23 of the 26 testing positive for the transgene. PCR primers designed to detect (a) the transgene, (b) the desired integration site, and (c) the site without integration, were developed by using targeted locus amplification (TLA, Cergentis®). These assays enabled identification of homozygosity for the desired transgene location. Of the 23 transgenic offspring produced, five were homozygous for the desired transgene location. This represents an application of OSD, coupled with ova transfer, for the production of homozygous offspring using poor quality cryopreserved semen to expedite herd propagation from a hemizygous founder transgenic male.