The production of cloned animals is, at present, an inefficient process. This study focused on the fetal losses that occur between Days 30-90 of gestation. Fetal and placental characteristics were studied from Days 30-90 of gestation using transrectal ultrasonography, maternal pregnancy specific protein b (PSPb) levels, and postslaughter collection of fetal tissue. Pregnancy rates at Day 30 were similar for recipient cows carrying nuclear transfer (NT) and control embryos (45% [54/120] vs. 58% [11/19]), although multiple NT embryos were often transferred into recipients. From Days 30-90, 82% of NT fetuses died, whereas all control pregnancies remained viable. Crown-rump (CR) length was less in those fetuses that were destined to die before Day 90, but no significant difference was found between the CR lengths of NT and control fetuses that survived to Day 90. Maternal PSPb levels at Days 30 and 50 of gestation were not predictive of fetal survival to Day 90. The placentas of six cloned and four control (in vivo or in vitro fertilized) bovine pregnancies were compared between Days 35 and 60 of gestation. Two cloned placentas showed rudimentary development, as indicated by flat, cuboidal trophoblastic epithelium and reduced vascularization, whereas two others possessed a reduced number of barely discernable cotyledonary areas. The remaining two cloned placentas were similar to the controls, although one contained hemorrhagic cotyledons. Poor viability of cloned fetuses during Days 35-60 was associated with either rudimentary or marginal chorioallantoic development. Our findings suggest that future research should focus on factors that promote placental and vascular growth and on fetomaternal interactions that promote placental attachment and villous formation.

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Dry, nonpregnant, mature cows (greater than 10 yr) of five breeds (Angus, A; Brahman, B; Hereford, He; Holstein, Ho; and Jersey, J) and their crosses (n = 60) were used in a 428-d experiment to determine maintenance energy requirements and efficiency of energy exchange. Cows were fed individually (via Calan electronic gates) a 70% cottonseed hull diet for four consecutive periods (127, 105, 97 and 99 d) at each of four levels (50, 83, 117 and 150% of each animal's estimated maintenance requirement). Each of four cows/breed group was assigned to one of the four feeding levels each period, with one cow fed each level each period. Body composition was measured initially and following each period in all cows via D2O dilution with a two-pool kinetics model procedure. Average ending live weight and empty body weight, protein and fat were similar to beginning values, indicating that cows began and ended in similar body composition. Dry matter digestibility (DMD) following the last period averaged 54.7%. Average DMD was 53.5, 57.8, 52.0, 55.0 and 51.7% (standard error of mean [SE] = 2.1) for A, B, He, Ho and J; values for He and J were lower (P less than .05) than for B. Diet digestible energy (DE) was similar for all breed types and averaged 62.4% of gross energy (GE). A small decrease in DE with increasing GE intake was noted for all breed types. The daily metabolizable energy requirement for weight equilibrium for A, B, He, Ho and J differed (P less than .01) and was 100, 98, 108, 119 and 152 kcal/kg.75 (SE 4.8), respectively, with an overall mean of 107. The ME for maintenance (MEm) was 91.6, 93.8, 95.3, 115.7 and 140.4 kcal/kg.75 for A, B, He, Ho and J (SE 6.0), respectively, with an overall mean of 101.9. Efficiency of weight change for A, B, He, Ho and J differed (P less than .01) and was 116, 135, 80, 116 and 58 g/Mcal ME intake, respectively, with an overall mean of 96. The respective efficiency of ME use for tissue energy gain or loss was 80.6, 66.8, 66.0, 36.5 and 36.2% for A, B, He, Ho and J, with an average energetic efficiency of 60.1%. In general, maintenance requirements for weight and energy equilibrium were lower in beef breeds and their crosses than in dairy breeds and their crosses. Efficiency of ME use also favored the beef breeds over the dairy breeds.(ABSTRACT TRUNCATED AT 400 WORDS)

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The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases.