Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A.

BACKGROUND: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. OBJECTIVES: To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. PATIENTS/METHODS: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. RESULTS AND CONCLUSIONS: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

Carbohydrate fermentation and nitrogen metabolism of a finishing beef diet by ruminal microbes in continuous cultures as affected by ethoxyquin and(or) supplementation of monensin and tylosin.

Long-term feedlot studies have shown positive effects (i.e., improved ADG and reduced morbidity and mortality) of dietary supplementation with ethoxyquin (AGRADO). This may be due to improving the antioxidant capacity at the ruminal, postruminal, or postabsorption levels. This study was designed to investigate the role of ethoxyquin at the rumen level. A finishing diet (12.5% CP; DM basis) was formulated to contain (on a DM basis) 77.5% flaked corn, 10% corn cobs, 10% protein/vitamin/mineral supplement, and 2.5% tallow. In a randomized complete block design experiment, the treatments were arranged as a 2 x 2 factorial. The main factors were two ethoxyquin treatments (without or with 150 ppm) and two monensin/tylosin treatments (without or with monensin and tylosin at 0.0028 and 0.0014% of dietary DM, respectively). Eight dual-flow, continuous culture fermenters were used in two experimental periods (blocks; 8 d each with 5 d for adjustment and 3 d for sample collection) to allow for four replications for each treatment. No interactions (P > 0.05) were detected for any of the measurements evaluated. Therefore, results of the main factors were summarized. Ethoxyquin supplementation improved (P < 0.05) true digestibility of OM (from 38.8 to 45.0%) but it did not alter (P > 0.05) concentrations of total VFA (averaging 131 mM) or acetate (averaging 58.8 mM). Ethoxyquin decreased (P < 0.05) propionate concentration from 51.1 to 42.4 mM and increased (P < 0.05) butyrate concentration from 18.4 to 22.9 mM. Digestion of total nonstructural carbohydrates was not altered (P > 0.05) by the treatments and averaged 86%. With the exception of increased (P < 0.05) concentration of propionate (from 42.0 to 51.5 mM) and decreased (P < 0.05) concentration of butyrate (from 25.9 to 16.3 mM), no effects (P > 0.05) were detected for monensin/tylosin. Ruminal N metabolism, including efficiency of bacterial protein synthesis (averaging 21.2 g N/kg OM truly digested), was not affected (P > 0.05) by the treatments. Results suggest positive effects of ethoxyquin on ruminal digestion of OM and unique changes in VFA production.

Induction of stable prenatal tolerance to beta-galactosidase by in utero gene transfer into preimmune sheep fetuses.

The successful transduction of hematopoietic stem cells and long-term (28 months) transgene expression within the hematopoietic system following the direct injection of high-titer retroviral vectors into preimmune fetal sheep was previously demonstrated. The present studies extended these analyses for 40 months postinjection and evaluated whether the longevity of transgene expression in this model system was the result of induction of prenatal tolerance to the transgene product. The intraperitoneal injection of retroviral vectors into preimmune sheep fetuses transduces thymic epithelial cells thought to present antigen and thus define self during immune system development. To directly demonstrate induction of tolerance, postnatal sheep were boosted with purified beta-galactosidase and showed that the peripheral blood lymphocytes from in utero-transduced sheep exhibited significantly lower stimulation indices to transduced autologous cells than did control animals and that the in utero-transduced sheep had a reduced ability to mount an antibody response to the vector-encoded beta-galactosidase protein compared with control sheep. Collectively, our results provide evidence that the direct injection of retroviral vectors into preimmune sheep fetuses induces cellular and humoral tolerance to the vector/transgene products and provide an explanation for the duration and stability of transgene expression seen in this model. These results also suggest that even relatively low levels of gene transfer in utero may render the recipient tolerant to the exogenous gene and thus potentially permit the successful postnatal treatment of the recipient. (Blood. 2001;97:3417-3423)

Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero.

Although promising, clinical and experimental efforts at in utero hematopoietic stem cell (HSC) transplantation currently are limited by minimal donor cell engraftment and lack of early donor cell expression after transplantation. We reasoned that cotransplantation of stromal elements (ST) might condition the fetal microenvironment for the engraftment of donor HSC and facilitate precocious bone marrow (BM) hematopoiesis. In this study we cotransplanted sheep ST, derived from adult or fetal BM, with either adult or fetal HSC, into preimmune fetal sheep. We analyzed donor cell chimerism in BM and peripheral blood and compared levels of chimerism achieved with recipients of HSC alone. In all experimental groups, stromal cotransplantation markedly increased the level of peripheral blood donor cell expression at 60 days after transplantation relative to controls. Adult BM-derived stroma cotransplanted with adult HSC provided the highest levels of circulating donor cells, whereas fetal-derived stroma was less effective. In addition, ST cotransplantation resulted in increased donor cell engraftment in the BM and led to significantly increased levels of donor hematopoiesis for over 30 months after transplant. Cotransplantation of stroma may represent a valuable clinical strategy for optimal application of in utero HSC transplantation.

The role of animal science in natural resource management: current decision making models and future needs.

Sustainable systems for land and natural resource management must be biologically, economically, and socially sustainable. Scientists and educators have historically viewed their role as developing new knowledge and technology to enhance biological and economic sustainability. Scientists have traditionally viewed sociopolitical sustainability and policy development as beyond our appropriate roles. Changing public values and perceptions on appropriate land use and natural resource protection are forcing land grant universities and their scientists and educators to re-evaluate traditional paradigms. The animal science community, in concert with the social and other biological sciences, must become more proactive in decision making processes on appropriate land use and natural resource management if we are to remain relevant. This paper describes the current situation and outlines approaches for the research and education communities to be important contributors to collaborative decision making processes on land and natural resource management.

Production by ewes rearing single or twin lambs: effects of dietary crude protein percentage and supplemental zinc methionine.

Eighty Targhee ewes with single or twin lambs were used to investigate the effects of chelated zinc methionine and level of dietary CP on milk and wool production, DMI, and ewe and lamb BW change. Treatments were arranged as a 2 x 2 x 2 factorial. Dietary treatments started 30 d prepartum and continued until 42 d postpartum. Milk production was estimated at 4, 10, 18, and 28 d postpartum. During gestation, DMI by ewes receiving zinc methionine and the 14.9% CP diet was greater (P < .10) than DMI by ewes not supplemented with zinc methionine and ewes fed the 11.3% CP diet. Ewes fed the 14.9% CP diet tended (P < .14) to gain more BW from 30 d prepartum to 4 d postpartum and lose more BW from 4 to 42 d postpartum than ewes fed the 11.3% CP diet. Ewes fed zinc methionine and the 14.9% CP diet produced more (P < .10) milk on d 28 than their counterparts. Ewes rearing twins produced more (P < .05) milk on d 4, 10, and 18 than ewes rearing single lambs. However, milk production on d 28 was not affected by number of lambs reared (P = .68). Lamb ADG at 28, 42, and 59 d postpartum was greater (P < .05) for both the zinc methionine treatment and the 14.9% CP diet. Zinc methionine (P = .11) and the 14.9% CP diet (P = .02) increased weaning weights by 6 and 9%, respectively. We conclude that both the zinc methionine supplement and the 14.9% CP diet resulted in more persistent milk production. In addition, when ewes and lambs were fed for an average of 42 d during early lactation, a 14.9% CP diet, and to a lesser degree, a chelated zinc methionine supplement, lamb weaning weights were improved.

Meat goat production and marketing.

Production opportunities, management strategies, and marketing options for meat goats in the United States are reviewed in this manuscript. The basis for any expansion must be goat production systems that are biologically and economically sustainable, meeting both producer and consumer needs. Meat goats historically have been kept for brush control. Their use to control noxious plants and in vegetation management will continue to be their primary role in the future. Meat goats are rarely the primary animal production enterprise in the United States, but they are becoming increasingly important contributors to the income of many producers. Meat goat marketing is highly unstructured in the United States, yet prices are generally higher on a per unit of weight basis than other red meat-producing species. Efforts to organize marketing have had only limited success. Over 90% of the world's goats are in developing countries. Goats are increasingly important in these countries as subsistence food producers. Production systems range from goats being a part of nomadic multispecies herds on arid desert rangelands, in agropastoral production systems, to goats being the primary animal enterprise in smallholder farming systems.

Carcass characteristics and optimal slaughter weights in four breeds of sheep.

Wether lambs from four breeds, Rambouillet (n = 38), Targhee (46), Columbia (38), and Polypay (38), were used to estimate an optimal slaughter weight for lean lamb production. Wethers, weaned at an average of 105 d of age and 38.5 kg, had ad libitum access to a finishing diet of 85% barley grain and 15% pelleted alfalfa and were slaughtered at weights ranging from 39 to 70 kg. Fat, protein, moisture, and ash percentages were determined on fat and lean from 7-rib racks. Optimal slaughter weight was estimated by using carcass backfat depth or extracted fat in the rack as dependent variables in linear equations. An acceptable lean lamb carcass was defined to have 2.5 to 6.5 mm of backfat depth and 26 to 40% rack chemical fat. On a weight-constant basis Columbia lambs were less physiologically mature than the other breeds, having less (P < .05) kidney and pelvic fat and a lower rack fat percentage. Targhee, Rambouillet, and Polypay wethers were similar in carcass fat and protein composition at a constant slaughter weight. Optimal slaughter weights for certified lean lamb carcasses of Targhee, Rambouillet, and Polypay wethers were 45 to 47 kg, resulting in a Choice quality grade and a 2.7 yield grade. The optimal weight for Columbia was heavier, between 45 and 55 kg, producing a Choice quality grade and a 2.4 yield grade. The derived optimal slaughter weights are limited to the populations sampled and may not be specific to other populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of simulated photoperiod alteration and induced estrus on reproductive performance of spring-born Columbia and Targhee ewe lambs.

The objective of this study was to test the hypothesis that a combination of induced estrus (IE) and melatonin (MEL) would increase reproductive performance in breeds characterized by late maturity more than either IE or MEL alone. Spring-born Columbia (C; n = 161; 188 to 222 d range of age at breeding; 49 to 80 kg range of BW at breeding) and Targhee (T; n = 166; 183 to 210 d range of age at breeding; 40 to 68 kg range of BW at breeding) ewe lambs were stratified randomly by breed to treatments: ambient controls (AC); IE; MEL; and MEL+IE. Melatonin (18 mg of Regulin) was implanted on September 7. Estrus induction included medroxyprogesterone acetate (MAP) pessaries inserted for 12 d and pregnant mare's serum gonadotropin (PMSG; 500 IU i.m.) at time of MAP removal. Ewe lambs were placed into two pens with five rams per pen by breed. Targhee ewes lambed at an increased rate with IE vs no IE (84.5 vs 67.9%; P < .01) and tended to lamb at an increased rate with MEL vs no MEL (82.5 vs 70.6%; P = .07). The 92.7% lambing rate observed in the Targhee ewe lambs for the combined treatment (MEL+IE) was slightly higher than an additive effect but the interaction between IE and MEL was not significant (P = .24). By contrast, none of the treatments altered (P = .5) lambing rate, number of lambs born, or number born alive for Columbia ewe lambs.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of barley variety and restricted versus ad libitum intake on rate, site, and extent of digestion by wethers fed a high-energy diet.

Twelve ruminally cannulated wethers (four with duodenal cannulas) were used in a replicated 4 x 4 Latin square to investigate effects of barley variety and restricted vs ad libitum intake on site, rate, and extent of digestion. Diets were based on Ottus (52.7% starch; O) or Steptoe (56.7% starch; S) barley fed at either 90% of prestudy ad libitum feed intake (R) or ad libitum consumption (A). Diets were formulated to be equal in starch content (38%). Periods consisted of 10 d for adaptation followed by 10 d in which fecal, duodenal, ruminal, and in situ collections were made. With the exception of ruminal starch digestion, barley variety did not interact with level of feed intake (P > .26). Intake of OM was 1,009, 996, 1,124, and 881 g.wether-1.d-1 for O, S, A, and R, respectively. Intake by R wethers of OM and starch was 78% (P < .01) of intake by A wethers. Duodenal flow of OM and ruminal pH were not affected (P > .28) by either intake restriction or barley variety. Although restricted intake did not affect ruminal (P = .84) and duodenal (P = .39) ammonia nitrogen, the O barley had higher (P = .01) ruminal and duodenal ammonia nitrogen than did S barley. Restricted intake had no effect (P > .30) on rate of ruminal barley digestion, ruminal and total tract starch digestion, or total tract OM digestion. This may be due to the rapid digestion rate of barley.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of different methods of estimating fecal output on plasma cortisol, fecal output, forage intake, and weight change in free-ranging and confined wethers.

Thirty-four white-face wethers (avg BW 56 kg) were used in two experiments to investigate differences in plasma cortisol, forage intake, fecal output, and BW change associated with two methods of estimating fecal output. The methods were fecal collection bags (B) and marker estimates of fecal output (U). A control treatment (C) was included in each experiment in which wethers were not bagged or dosed with the marker. Experiment 1 was conducted with wethers grazing dormant sagebrush-bunchgrass range. Experiment 2 was conducted with wethers housed in metabolism crates. In Exp. 1, the following differences were detected: 1) basal and mean integrated (following ACTH challenge) plasma cortisol levels were higher (P < .05) for U wethers than for C wethers, 2) no differences (P > .05) between B and C wethers were detected for basal or mean integrated plasma cortisol, 3) both B and U wethers gained (P < .05) weight, whereas C wethers lost weight, and 4) no differences (P > .05) between B and U wethers were detected for cortisol measurements, weight change, or fecal output. No differences (P > .05) were noted for any of the variables tested in Exp. 2. When treatments were combined within experiment to test Exp. 1 vs 2, time to peak cortisol levels was longer (P < .05) and mean integrated cortisol levels and weight change were lower (P < .05) for wethers in confinement than for wethers on range. No difference (P > .05) was detected for basal cortisol levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of diet composition and feed intake level on diet digestibility and ruminal metabolism in growing lambs.

Lambs (29 +/- 2.5 kg) were fed three diets at various intakes to determine whether diet composition or level of intake was reflected in changes in diet digestibility or ruminal fluid characteristics. In Exp. 1, a 90% concentrated, pelleted diet or a whole shelled corn diet with a pelleted protein supplement was fed at three levels of intake: ad libitum and 92.5 and 85% of ad libitum (n = 15). Exp. 2 compared the 90% concentrate diet with diets in which the energy density was diluted to 55 or 72.5% concentrate by including alfalfa hay as a possible method of restricting energy intake (n = 6). Lambs were adapted to diets for 13 d; feces were collected for 6 d and ruminal fluid was collected 0, 3, and 6 h after feeding on the day following fecal collection. Restricting intake in Exp. 1 did not affect DM digestibility or digestibility of CP or starch. Digestibility of ADF was increased (P less than .10) by restricting intake. Ruminal fluid pH, ammonia concentration and VFA concentrations were affected little by either restricted intake level. Digestibility of DM was 4% higher (P less than .001) and starch 5% higher (P less than .001) for the whole shelled corn diet than for the pelleted, high-concentrate diet. Ruminal pH of lambs fed the whole shelled corn diet was higher and fluctuated less than the ruminal pH of lambs fed the high-concentrate, pelleted diet. In Exp. 2, diet digestibility was reduced (P less than .01) and ruminal pH was increased (P less than .002) by addition of hay. Restricted feeding of lambs did not seem to increase diet digestibility or alter ruminal conditions.

Effect of level of intake and supplemental barley on marker estimates of fecal output using an intraruminal continuous-release chromic oxide bolus.

Sixty wethers (average BW = 45 kg) fitted with fecal collection bags were used in four experiments to evaluate the ability of a intraruminal continuous-release chromic oxide bolus to predict fecal DM output. In Exp. 1, 18 wethers housed in metabolism crates were fed barley at either 0, 100, or 200g/d and allowed ad libitum consumption of alfalfa pellets. In Exp. 2, 18 wethers were housed in metabolism crates and fed alfalfa pellets at either 70, 85, or 125% of pre-study ad libitum consumption. In Exp. 3, 12 wethers grazed a sagebrush-bunchgrass range and were individually fed barley at 0 or 200 g/d. In Exp. 4, 12 wethers grazed either an ungrazed (383 kg/ha herbaceous biomass) or a heavily grazed (175 kg/ha herbaceous biomass) sagebrush-bunchgrass range. Experiments 1 and 2 were balanced 3 x 3 Latin squares, and Exp. 3 and 4 were crossover designs. Chromium content was determined in rectal grab samples. Treatment effects were compared using marker-estimated fecal output divided by total fecal collection, multiplied by 100, as the dependent variable. Accuracy of the estimate was verified by comparing marker-estimated fecal output with total fecal collection using a paired t-test. In Exp. 1 and 2, treatments were different (P less than .05). No differences (P greater than .50) were detected in Exp. 3 and 4. In Exp. 1, 2, and 3 accuracy was different (P less than .02) among wethers within study and treatment. Only in Study 4 were minor or no differences (P greater than .09) in accuracy found.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of breed, number of suckling lambs, and stage of lactation on ewe milk production and lamb growth under range conditions.

Milk production and lamb growth were characterized in 118 multiparous, 3- to 7-yr-old Rambouillet, Columbia, Polypay, and Suffolk ewes under spring sage range and high mountain meadow grazing from 28 to 98 d postpartum. Daily milk yield as measured by the lamb suckling weight differential technique did not differ (P greater than .05) among breeds, although milk production of Suffolk ewes tended to be higher than that of the other three breeds. Within the Rambouillet, Columbia, and Polypay breeds, total estimated yield of ewes with twins was 13 to 17% higher than that of ewes with singles, whereas in the Suffolk breed, suckling twins increased total milk yield 61% over that of ewes with singles. Twin lambs induced a larger differential in dam milk production in late lactation (70 to 98 d) than in earlier lactation (28 to 70 d). Number of lambs did not influence milk protein, Ca, or P content (P greater than .05). Fat levels in colostrum and 4-d milk were elevated 14 and 20%, respectively, in ewes suckling twins compared with ewes suckling singles. Under range conditions, Suffolk ewes suckling single or twin lambs lost more BW (12 and 21% of 4-d postpartum body weight, respectively) than Rambouillet (4 and 7%), Columbia (5 and 8%), or Polypay (8 and 8%) ewes. Correlation coefficients of milk production and lamb growth rate were positive and significant (P less than .05) up to 56 d of age. Growth rate was less closely associated with milk production for twin than for single lambs.

Simulation of lean lamb production systems.

A series of simulations were performed to evaluate the effects of mature size (WMA; 60, 70, 80 and 90 kg), slaughter weight (SW; 55, 60 and 65 kg) and three diets fed to feedlot lambs. Rations included a traditional diet (C2L) or placing weaned lambs on alfalfa for either 30 (A30) or 60 (A60) d before placing them on the C2L diet (A30-C2L and A60-C2L). Two lamb pricing schemes were used in the financial analysis of the biological results: sale price based on weight with or without adjustment for carcass fat. Placing lambs on alfalfa pasture increased ownership by 22 d on average compared to C2L. As WMA and SW increased, ownership increased from 3 to 11 d. Across nutritional treatments, fat decreased by 5.8 to 9.0% as WMA increased from 60 and 90 kg. When market lambs were priced on weight, net returns per lamb were greatest for A30-C2L or A60-C2L ($2.34 and $5.21, respectively above C2L) and for greater SW ($4.77 and $7.16 greater for SW 60 and 65 kg than for SW 55). The combination WMA of 90 kg, a SW of 65 kg and nutritional treatment of A60-C2L proved most profitable. However, when sale price was discounted for excessive fat content, the combinations WMA of 60 or 70 with a SW of 65 and A60-C2L were most profitable. The rank correlation between biological efficiency (weight gain/dry matter feed consumption) and net return, when sale price was based on weight, was found to be -.51. This was because lambs slaughtered at lighter weights had high biological efficiencies but low net returns due to a negative price margin. With negative price margins but positive feeding margins, net returns are greatest for heavier, fatter lambs fed longer.

Prevalence and effect of subclinical ovine progressive pneumonia virus infection on ewe wool and lamb production.

The prevalence of infection with ovine progressive pneumonia (OPP) virus and its effects on ewe wool and lamb production were investigated in a flock of 2,976 ewes of 6 breed types (Rambouillet, Targhee, Columbia, Polypay, 1/4 cross Finnsheep, and 1/2 cross Finnsheep). Prevalence of seropositivity was significantly (P less than or equal to 0.01) lower among Rambouillet and Targhee breeds (44 and 42%, respectively), intermediate in Polypay, Columbia, and 1/4 cross Finnsheep (approximately 53%), and higher among 1/2 cross Finnsheep (62%). Seropositivity increased with age in all breed types from 11% at 1 year of age to 93% at greater than or equal to 7 years of age. Lateral disease transmission is indicated by linear increase of seropositivity prevalence with increasing age, including that in sheep greater than 6 years old. Subclinical infection with OPP virus had no apparent detrimental effect on number of lambs born, lamb viability, birth weight, number of lambs weaned, or growth rate of single and twin lambs, compared with findings for noninfected sheep in the same flock. Mature ewe body weight and grease fleece weight did not differ between subclinically infected seropositive and seronegative ewes. Subclinical infection with OPP virus does not appear to have an adverse economic effect on ewe wool and lamb production. Culling rate attributable to clinical manifestation of infection with OPP virus must be accurately determined before the true effects of virus infection on production can be determined and an eradication program can be recommended.

Effect of altering nutrient density (concentrate to roughage ratio) and restricting energy intake on rate, efficiency and composition of growing lambs.

Two hundred ninety-eight Rambouillet ewe and wether lambs approximately 9 mo old from Texas were allotted to pens (29 to 30 lambs/pen). Two replicate pens were fed each of five diets. Two methods of restricting energy intake were evaluated: altering nutrient density (55% vs 72.5% vs 90% concentrate levels) and restricting intake of the 90% concentrate pelleted diet (92.5% or 85% ad libitum intake). Daily gains were highest with the 72.5% concentrate level and feed efficiency was poorest with the 55% concentrate diet fed ad libitum. Altering nutrient density did not reduce energy intake because lambs compensated by increasing feed intake at the lower nutrient densities. Actual feed intake was 89.5% of ad libitum for the 92.5% of ad libitum intake level and 84% of ad libitum at the 85% of ad libitum intake level due to refusal to consume some fines from the 90% concentrate diet. Feed refusal was greatest for lambs with ad libitum access to feed. Average daily gain was depressed by 8% at the 85% intake level. However, feed efficiency was improved (P less than .01) by 20% when intake was restricted to 92.5% of the ad libitum intake level. Differences in carcass traits were small, but consistent; carcass fat content was reduced slightly by restricting feed intake at constant slaughter weight.