To date, the efficiency of pig cloning by nuclear transfer of somatic cell nuclei has been extremely low, with less than 1% of transferred embryos surviving to term. Even the utilization of complex procedures such as two rounds of nuclear transfer has not resulted in greater overall efficiencies. As a result, the applicability of the technology for the generation of transgenic and cloned animals has not moved forward rapidly. We report here a simple nuclear transfer protocol, utilizing commercially available in vitro-matured oocytes, that results in greater than 5% overall cloning efficiency. Of five recipients receiving nuclear transfer embryos produced with a fetal fibroblast cell line as nuclear donor, all five established pregnancies by day 28 (100%), and 4/5 (80%) went to term. Efficiencies for each transfer were 7% (9 piglets/128 doublets transferred), 5% (5/100), 12% (7/59), and 6.6% (7/106). The overall efficiency in all recipients was 5.5% and in pregnant recipients 7.7%, with a total of 28 cloned piglets produced. With the average fusion rate being 58%, the percentage of fused doublets producing a live piglet approached 12%. The method described here can be undertaken by a single micromanipulator at a reasonable cost, and should facilitate the broad utilization of porcine cloning technology in transgenic and nontransgenic applications.

Jersey x Holstein crossbreds (JxH; n = 76) were compared with pure Holsteins (n = 73) for 305-d milk, fat, and protein production; conception rate; days open; proportion of cows pregnant within fixed intervals postpartum; and body and udder measurements during first lactation. Cows were housed at 2 research locations of the University of Minnesota and calved from September 2003 to May 2005. The JxH were mated to Montbeliarde sires, and Holstein cows were mated to Holstein sires. Best Prediction was used to determine actual production (milk, fat, and protein) for 305-d lactations with adjustment for age at calving, and records less than 305 d were projected to 305 d. The JxH (274 kg) and pure Holsteins (277 kg) were not significantly different for fat production, but JxH had significantly less milk (7,147 vs. 7,705 kg) and protein (223 vs. 238 kg) production than pure Holsteins. The JxH had significantly fewer days open than pure Holsteins (127 vs. 150 d). Also, a significantly greater proportion of JxH were pregnant at 150 and 180 d postpartum than pure Holsteins (75 vs. 59% and 77 vs. 61%, respectively). The JxH had significantly less body weight (60 kg) at calving, but significantly greater body condition (2.80 vs. 2.71). Furthermore, JxH had significantly less udder clearance from the ground to the bottom of the udder than pure Holsteins (47.7 vs. 54.6 cm), and greater distance between front teats (15.8 vs. 14.0 cm) than pure Holsteins during first lactation.

Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection of Mycobacterium avium subsp. paratuberculosis from milk and/or fecal samples from cattle and American bison. Samples were spiked with M. avium subsp. paratuberculosis organisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detect M. avium subsp. paratuberculosis in feces and milk. By use of this simple and rapid technique, 10 or fewer M. avium subsp. paratuberculosis organisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and costeffective for the diagnosis of clinical and subclinical Johne's disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions.

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The objective of the present study was to determine differences in time of detection of pregnancy between heifers and cows and the interval after insemination at which the maximum sensitivity and negative predictive value of transrectal ultrasonography were obtained. One-thousand-four-hundred transrectal ultrasonographies (TRUS-1; 1,079 in cows and 321 in heifers) were performed using a 5-MHz linear-array transducer. The cattle were randomly assigned to have TRUS performed once between days 24 and 30 (estrus=day 0) in cows or between days 21 and 27 in heifers. Every TRUS diagnosis was subsequently compared with a second TRUS diagnosis (TRUS-2), performed 3-8 days later, after day 30 (range 31-38) for cows and after day 27 (range 28-35) for heifers. The sensitivity and specificity between cows and heifers for the common days of TRUS (from 24 to 27) were compared. In cows, sensitivity increased gradually from 74.5% at day 24 to 100% at day 29 (P<0.01). Specificity increased from days 24-25 and reached a plateau of 96.6% on day 26 (P<0.01). In heifers, sensitivity increased from 50% at day 21 to 100% at day 26 (P<0.01). Specificity increased from 87.5% at day 21 and remained steady at 94% starting on day 23 (P>0.05). The sensitivity for cows and heifers was 89.2 and 96.8%, respectively (P<0.05) and the specificity was 93.0 and 93.4% (P>0.05). In this study, heifers were diagnosed pregnant earlier than cows, and the maximum sensitivity and negative predictive value were obtained 3 days earlier in heifers than cows (days 26 and 29, respectively).