41 TRANSMISSION OF A PARASITE-RESISTANT PHENOTYPE FROM A DONOR RAM TO ITS CLONED OFFSPRING

Abstract

A 9-year-old parasite-resistant superfine Merino ram was cloned to study the inherited phenotype in cloned offspring. This donor ram was >3 standard deviations below the mean for internal parasite fecal egg counts in a 10 000 sheep flock. Two viable rams were produced from 3 days of NT. Their health status was studied from birth to maturity and their fertility and progeny via an AI program. Their mucosal immune system was indirectly assessed through their ability to cope with high internal parasite challenges. Oocytes were extracted from abattoir ovaries in late autumn. Serum-starved fibroblasts were combined with enucleated oocytes and fused; the couplets wereactivated at 25–26 h postmaturation with ionomycin and then DMAP. Embryos were cultured in a 2-stage bovine embryo culture medium (Cook) for 6 days in 5% oxygen. From 3 days of NT, all 37 embryos reaching blastocyst or compact morula stages were transferred into 14 ewes. At Day 30, 6/14 ewes were pregnant with single fetuses and 1 fetus died by Day 60. Each of the 5 lambs had breathing difficulties at birth and 3 died. For 2 weeks prior to term, maternal serum levels (collected every other day) of progesterone and estradiol were not different between viable and non-viable pregnancies. In the 2 viable pregnancies, cortisol levels peaked (>10 ng mL^–1), then dropped sharply (<2 ng mL^–1 for 4 days), and then rose to the time of birth (>7 ng mL^–1). This cortisol profile may help explain why the 2 viable lambs acquired sufficient lung capacity by the time of birth to survive. Samples for fecal egg counts and complete blood counts were collected monthly for 12 months on the 2 surviving ram lambs. Fecal egg counts were 2–5 times higher than for 2 control animals, with peak counts reaching 3000–4000 eggs per gram. Larval culture showed the eggs were predominantly Hemonchus. Clone 2 had consistently higher egg counts than clone 1. Both clones had persistent loose feces for the first 6 months and Clone 2 continues to have diarrhea. Red blood cell counts were consistently low normal (8–11 million/µL). White cell counts were normal, other than when high counts (16 000 per µL) corresponded with high fecal egg counts. Body weight, wool quantity, and fiber diameter at 1 year of age were slightly different between clones (59 kg; 5.3 kg; 16.4 µm vs. 54 kg; 4 kg; 16 µm, respectively). Scrotal circumference was larger for clone 1 than for clone 2 (29 cm vs. 24.5 cm). Semen from the rams was cryopreserved for use in an AI program (50 ewes per sire) to progeny-test lambs produced by the 2 rams, the original donor ram, and a link ram, with lambing due in October 06. Overall, clone 1 has remained consistently healthier than clone 2. High fecal egg counts indicated significant internal parasite challenge during their first year of life, which resulted in elevated white blood cell responses. The obvious clinical effect of high egg counts was diarrhea, which may indicate a beneficial response to parasite infection. Continued studies will further document their parasite resistance, immune responses, and progeny characteristics.