In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.

The aim of this study was to hormonally induce lactation in prepubertal, nulliparous, and male goats both transgenic and non-transgenic. Analysis of milk quality, recombinant protein expression levels, total amount of recombinant protein produced, and the affect on long-term reproductive capability was assessed. Fifty-one goats (Saanen, Alpine, and Toggenburg), male and non-pregnant females, 2-31 months of age, either non-transgenic or transgenic were evaluated with a total of 10 transgenes (constructs) represented. Animals were given estradiol (0.25 mg/kg, i.m.) and progesterone (0.75 mg/kg, i.m.) on days 1, 3, 5, 7, 9, 11 and 13, while prednisilone (0.4 mg/kg, i.m.) was administered on days 14-16 with mammary massage occurring daily from day 5 onward. Forty of 51 animals, (36 of 38 females and 4 of 13 males) produced milk with total volumes in the 30-day experiment, ranging from 20 microl to 530 mls per day, or approximately 500 microl to 6.8 liters total. Milk composition was analyzed for various parameters (total protein, fat content, total solids and somatic cell count) with no significant differences found between induced and natural milk. Expression levels of recombinant proteins from transgenic animals that were analyzed during the induced lactation, and subsequently during normal lactations, were found to have no significant differences. Total amount of recombinant protein produced was evaluated at different expression levels with no statistical significance seen. While over 90% of the females placed in the regimen became pregnant, there was a correlation between increased age at time of induction and an increase in number of breedings, or reproductive cycles needed to establish a pregnancy after induction. For males, 100% placed in the regimen settled females after hormonal induction of lactation. Semen quality was evaluated prior to, during, and after hormonal treatments. Semen volume and sperm number did not differ; however, for a small percentage of males, there was a decrease in sperm and post thaw motility after hormonal treatments. These levels returned to normal within 4-5 weeks. Subsequent natural lactations showed total milk volumes within breed standards. These findings indicate that hormonal induction of lactation in the caprine species is a viable alternative to pregnancy for initiating lactation and milk production, does not adversely impact reproductive performance long-term, and can benefit the early assessment of recombinant proteins produced in a transgenic founder program.

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Production of human recombinant proteins in the milk of transgenic animals has been shown to be a viable production system. Protection of the animal genetics involved is paramount. Vitrification of embryos is a simple, time-efficient way of preserving an animal’s genetics without the formation of damaging ice crystals during the freezing process. Cytochalasin B has been shown to increase the viability of porcine blastocysts by reducing damage to microfilaments and other cytoskeletal components. These experiments utilized caprine parthenogenic blastocysts as a model to compare the viability of parthenotes treated with or without cytochalasin B prior to and during vitrification. Abattoir oocytes were in vitro-matured in M199 with 10% goat serum containing FSH, LH and gentamycin for 18 to 21h. Parthenogenic blastocysts were produced by treating in vitro matured abattoir oocytes with ionomycin for 5min (5μM) and with 6-dMAP (3mM) for 3h followed by culturing in SOF+0.8% BSA for 7 to 8 days at 38°C with 6% O2, 5% CO2, and 89% N2 in a modular incubator chamber. The experimental group was treated with cytochalasin B (5μg/mL)in the culture media for 30 to 45min prior to and thereafter throughout the vitrification process. All blastocysts (both the experimental group and the control group) were washed through two ovum culture media (OCM) droplets for 5min each. The blastocysts were incubated in vitrification solutions 1 and 2 (10% glycerol in OCM and 10% glycerol+20% ethylene glycol in OCM, respectively) for 5min each, followed by vitrification solution 3 (25% glycerol+25% ethylene glycol in OCM). They were then aspirated immediately into a 0.25cc cryopreservation straw, followed by an air bubble, and then a 0.25M sucrose solution in OCM. The straws were immediately plunged into liquid nitrogen and stored at −196°C. One to four days later, straws were thawed in air for 5s at room temperature, then in 22°C water for 15s. After thawing, the contents of the straw were expelled, mixed, held for 5min, and finally placed in OCM for 5min. Recovered embryos were placed in SOF+20% FBS and incubated at 38°C with 5% CO2 in air overnight. Viability was determined by re-expanding and subsequent hatching of the blastocyst. As shown in Table 1, there were no significant differences between re-expansion and hatching of blastocysts with cytochalasin B treatment compared to blastocysts not treated with cytochalasin B. These results suggest that, unlike porcine embryos (Dobrinsky et al., 2000 Biol Reprod 62, 564–570), cytochalasin B treatment does not improve the post-thaw viability of vitrified caprine parthenogenic blastocysts. Table 1

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