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Department of Animal Science
University of Minnesota
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Phone: 612-624-2722
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Swine Research Abstracts

2007

RNA interference of mouse CFTR function; towards a pig model of cystic fibrosis

D.F. Carlson^1,2, A.M. Geurts², M. Palmer-Densmore¹, C. Won Park³, W. Warwick⁴, C. Steer³, C. Milla⁴, S. O'Grady¹, D.A. Largaespada², and S.C. Fahrenkrug^1,2

Although mouse models of CF have been generated that manifest some of the electrophysiologic characteristics of CF, their benign pulmonary phenotype renders these models incomplete. Pigs may provide a superior model given the importance of CFTR in the transport of chloride, bicarbonate and fluid across their pulmonary mucosa. Pigs may also represent a better model for the development of gene therapeutics given that their bronchial branching, vascular distributions, and morphologic characteristics are strikingly similar to humans. We have undertaken the development of a porcine model of CF by transpositional transgenesis (TnT) with shRNA expressing transposons. As proof of principle, mice were created by pronuclear injection of a CpG methylated and nonmethylated Sleeping Beauty (SB) transposon driving the expression of a short hairpin RNA directed against the mouse CFTR mRNA (shCFTR). Mouse transgenesis was extremely efficient using the methylated transposon (90%), and was accompanied by erasure of transgene methylation and reliable expression from both pol II and pol III promoters. Several F1 animals with distinct TnT insertions displayed phenotypes consistent with CF, including impairment of chloride transport in colonic epithelia and impairment of reproduction.

In vitro CF models in cultured pig endometrial epithelial cells by TnT with analgous transposons reduced CFTR mRNA and chloride channel transport by 90-94%. Several transgenic pig fibroblast clones were developed as nuclear donors for somatic cell nuclear transfer (SCNT) and characterized for shRNA expression by quantitative PCR. A selected fibroblast cell line was cloned at the National Swine Resource and Research Center and resulted in the birth of 7 healthy piglets harboring the same transgene insert locus. While the presence of the transposon and expression of a Pol II transcript was confirmed by PCR from ear and tail biopsy, expression of the shCFTR hairpin was not detected. Founder pigs will be sacrificed and characterized for shRNA expression and levels of CFTR mRNA in relevant tissues. Alternatively, our remarkable transgenesis rate at multiple loci and accurate recapitulation of the CF phenotype in mice provides a compelling argument for production of founder pigs by pronuclear TnT.

¹Animal Biotechnology Center and Department of Animal Science, University of Minnesota
²Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota
³Department of Medicine, University of Minnesota
⁴Department of Pediatrics, University of Minnesota

A toolbox for transgenesis and functional annotation of the porcine genome

K.J. Clark^1,2, D.F. Carlson^1,2, L.K. Foster¹, A.R. Bents¹, and S.C. Fahrenkrug^1,2

The swine genome-sequencing initiative has been undertaken to aid in the annotation of a species with importance to both animal agriculture and biomedicine. Comparative information from human and model organism genomes will provide useful information for annotating the function of pig genes. However, the function of the majority of vertebrate genes remains to be determined. Transposons have historically played an important role in the annotation of invertebrate and plant genomes by transgenesis and insertional mutagenesis. For transgenesis, transposons offer an efficient and precise mechanism for the integration of DNA that avoids incorporation of CG-rich vector DNA and multi-copy transgene concatemerization that can lead to suppression of gene expression. For insertional mutagenesis, transposons tag mutated loci, facilitating the isolation and annotation of affected genes. Recently, these tools have been applied to the annotation of mouse and zebrafish gene function at the Beckman Center for Transposon Research. We have also begun to apply transposons and recombinases to the manipulation and annotation of the porcine genome. We have developed and tested a transposon toolbox (including Sleeping Beauty, Tol2, piggyBac, and a novel transposon from P. platessa) in pig cells, observing up to a 50-fold increase in the rate of transgenesis. CRE and FLP recombinases have effectively been applied to conditional gene expression and selection-cassette recycling in pig cells. Strategies for the application of transposons and recombinases to swine transgenesis and functional gene annotation will be discussed.

¹Animal Biotechnology Center and the Department of Animal Science, University of Minnesota
²Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota

Injectable progesterone does not reduce mid to late pregnancy failure of sows during summer infertility

L.J. Johnston¹, E. Keetelaar², and R.J. Smits²

Reproductive failure is an important cause of culling sows. Pregnancy failure due to irregular returns to oestrus and non-pregnant sows at expected farrowing can account for 14 % of non-age based culling of sows. Social stresses are related to increased rate of pregnancy failure and may be mediated through a decline in plasma progesterone (P₄). Low P₄ may occur during seasonal infertility or when sows are relocated or mixed in groups after pregnancy diagnosis. Sows may benefit from exogenous P₄ during these stressful periods. Spanish researchers supplemented lactating dairy cattle with P₄ and reduced pregnancy loss. In an earlier experiment, we observed a trend for improved farrowing rate when sows received one intramuscular (i.m.) injection of 125 mg P₄ on day 42 postcoitum. These results required verification with a statistically valid sample size.

We hypothesized that a single injection of P₄ on day 34 post-mating would increase the proportion of sows remaining pregnant to full term when subjected to stresses associated with mixing social groups and environmental conditions during seasonal infertility. Large White x Landrace F₁ sows (n = 522) were selected randomly from matings conducted during late summer (Week 2 through 8) in a commercial production unit in Victoria. Sows were mated artificially and housed in individual stalls from weaning through 34 ± 1.5 days post-mating. On day 34 post-mating after positive diagnosis of pregnancy by ultrasound, sows were moved from stalls to group pens of 17-18 sows per pen (1.4 m²/sow). Treated sows received one i.m. injection of 125 mg P₄ (Jurox Progesterone) in the neck while control sows received no injection. Sows were housed in pens from day 34 post-mating until farrowing or the sow failed to farrow. Categorical data were analysed using Chi square. Treatment effects on litter size were analysed using analysis of variance with sow parity (range = 0 to 8) used as a covariate.

Progesterone injection did not affect the incidence of pregnancy failure after week five of gestation in this experiment (Table 1). Overall, 12.6 % of sows confirmed pregnant at five weeks of gestation failed to maintain pregnancy and typified seasonal infertility on this farm. There were no interactions between P₄ treatment and sow parity or week of mating for pregnancy loss. Litter size at farrowing was not affected by P₄ treatment. Distribution of litter sizes at subsequent farrowing (range = 3 to 18 live pigs) was not affected by P₄ treatment (χ2 = 3.94; P < 0.42).

We conclude that one i.m. injection of P₄ at the time of moving sows from stalls to group pens at week five of gestation does not reduce pregnancy loss during a time of seasonal infertility.

¹West Central Research and Outreach Center, University of Minnesota, Morris
²QAF Meat Industries, Corowa, New South Wales

Effects of familiarity and weight variation on aggression among grow-finish pigs following regrouping

Y.Z. Li and L.J. Johnston
West Central Research and Outreach Center, University of Minnesota, Morris

An experiment was conducted to study effects of familiarity and weight variation at regrouping on aggression and growth performance of pigs. Pigs (n = 180) at 8 wk of age (BW = 23.0 ± 3.11 kg) from 3 group farrowing rooms where they mingled within each room from 10 d of age were used. Five groups of 9 pigs (5 males:4 females) were allotted to each of the four treatments: familiar pigs of uniform BW, familiar pigs of mixed BW, unfamiliar pigs of uniform BW and unfamiliar pigs of mixed BW. Familiar groups consisted of pigs from one farrowing room, and unfamiliar groups consisted of 3 pigs from each of the 3 farrowing rooms. Uniform groups were formed by using the middle 2 quartiles and the mixed weight groups by using the heaviest and lightest quartiles of pigs. Aggression and activity were observed directly for 4 h during the first 3 d, on d 7, and d 14 post regrouping. Skin lesions on head, shoulders and body were scored (scale 0-3 based on the number of scratches) on all pigs 48 h post regrouping. Total fighting durations were higher in unfamiliar groups during the first (48.5 vs. 0.5 s/(pig.4h), SE = 15.39; P < 0.001) and second day (10.8 vs. 0.4 s/(pig.4h), SE = 4.58; P < 0.005) post regrouping than in familiar groups. Unfamiliar pigs had higher total injury scores (6.5 vs. 1.2, SE = 0.40; P < 0.001) and spent less time eating during the first day (5.2 vs. 8.9%, SE = 1.31 of total observation time; P < 0.01) post regrouping than familiar pigs. There was an interaction (P < 0.02) in injury score between familiarity and BW variation as pigs of mixed BW compared with uniform BW increased injury scores in unfamiliar groups but not in familiar groups. ADG in unfamiliar groups was lower during the initial 6 wk (809 vs. 849 g/d, SE = 11.2; P < 0.001) but not for the entire 15 wk (806 vs. 821 g/d, SE = 9.7; P = 0.11) compared with familiar groups. Familiarity did not affect ADFI or G:F. Sorting pigs by familiarity and weight at mixing could be beneficial in terms of alleviating aggression-associated injuries.

2006

Dietary supplementation of oregano essential oils (OEO) in gestation and lactation sow diets and its effect on reproductive performance of sows, growth pattern of piglets and their immune status

C. Ariza-Nieto, S.K. Baidoo, R. Walker, M.R. Hathaway, M. Bandrick, and T.W. Molitor
University of Minnesota, St. Paul

This study involved supplementation of OEO in gestation and lactation diets on sow reproductive and suckling pig performance. Twenty four hours after breeding, 144 sows were randomly assigned to control and oregano (OEO 250ppm, Regano500®). Blood was collected via jugular from 6 randomly chosen sows per treatment and their litter at farrowing (piglets bled before and after suckling), 7, and 14 days of lactation. Colostrum and milk samples were collected on these days. T lymphocytes were phenotyped (γδ, CD8, CD4) from whole blood. Natural cytotoxicity was used to assess innate immunity in pigs. To evaluate growth rates due to OEO supplementation, 630 piglets were individually identified and weighed at 1, 5, 9, 12, 16, and 19 days of age. The number of live born piglets was higher when sows were supplemented with OEO during gestation (+1.1 piglets, P<0.05). Sows fed OEO during gestation-lactation improved litter weaning weight and litter weight gain (2.7 and 3.3 kg; P<0.05) compared to control. Wean to service interval was lower in OEO fed sows compared to control (5 v 8 days). Growth rate of piglets was higher (P<0.01) in sows fed OEO on 1-5, 9-12, and 16-19 days of lactation. The percentage of γδ lymphocytes isolated from piglets before suckling from sows fed OEO was greater (P<0.05) than control, although no differences were observed after suckling. These pigs also demonstrated greater natural killer (NK) activity throughout lactation, and significantly greater NK activity before suckling (P<0.01). OEO supplementation during gestation and lactation shows a biological growth potential on suckling piglets.

Eating sequences of gestating sows in group housing with electronic sow feeders

Y.Z. Li^1,2 and H.W. Gonyou¹

Electronic sow feeders (ESF), a sequential feeding system, have been used in group housing of gestating sows to control individual intake and aggression during feeding. However, the effect of sequential feeding on sow eating behaviour and performance is not clear. A hypothesis was that gestating sows may adapt to sequential feeding systems by establishing a feeding order. An experiment was carried out to study the feeding order of gestating sows in group housing with ESF. A total of 146 sows in parity 1 to 6 were studied in seven static groups. Each of the three pens used was equipped with an ESF and accommodated 36±2.1 (SD) sows. Space allowance was 2.1 m2 per sow. Sows from two breeding groups were simultaneously moved into each pen, one group at 5 d (pre-implantation) and another at 40 d (post implantation) post breeding. All sows were limited fed according to their nutritional requirements. Feeding cycle started at 3 pm daily. Feeding order was recorded at d 3 and d 56 (wk 8) post mixing by the computer which controlled the feeding system. Feeding order score was calculated for each individual sow (Feeding order score = (p–1) / (n-1); where p = sow’s numerical position within the feeding order, n = total number of sows ate in the feeder). Weight gain during gestation and farrowing data (total born, live born, dead born) were collected. The results showed that 80-90% of sows entered ESF and ate their ration voluntarily on d 3 post mixing. Pre-implantation sows ate earlier than post-implantation (P < 0.01) and mature sows (parities 3 to 6) ate before sows in parity 1 and 2 (P < 0.05) in feeding order on d 3. On wk 8, neither sow parity nor gestation stage affected feeding order. There was not correlation in feeding order scores between day 3 and wk 8. Higher feeding order scores (ate later) did not affect farrowing performance and weight gain during gestation. These results suggest that gestating sows can not adapt to sequential feeding systems by establishing and maintaining a feeding order. Although reproduction performance was not affected by feeding order, sow competition for the feeder raised welfare concerns in sequential feeding systems.

¹Prairie Swine Centre, Saskatoon, Canada
²Current Address: West Central Research & Outreach Center, University of Minnesota, Morris

Factors related to piglet pre-weaning mortality in a bedded group farrowing system

Y.Z. Li, L.J. Johnston, and A.M. Hilbrands
West Central Research & Outreach Center, University of Minnesota, Morris

Recently there has been increased interest in loose farrowing systems in North America due to sow welfare concerns. However, piglet mortalities of 25 to 33% have been reported in these systems. We investigated factors related to piglet mortality in a group farrowing system. A total of 156 sows (Landrace x Yorkshire) in parity 0 to 5 and their litters were studied in three farrowing rooms. All sows were group housed during gestation, and moved to the farrowing room one wk before expected farrowing dates.  Each farrowing room (9.6 x 10.8 m) was equipped with eight straw-bedded, ‘get-away’ pens (2.4 x 3.0 m) to accommodate eight sows and their litters. A communal eating/drinking/dunging area was provided in each room.  The pens were removed about d 10 post farrowing, so sows and their litters mingled in a group.  Minimal cross fostering was conducted within 2 d of farrowing. Piglets were weaned at 28 to 36 d of age. The PROC MIXED procedure of SAS was used to analyze effects of sire breed (Yorkshire vs Landrace) of the sows, sire breed (Duroc, Yorkshire vs Landrace) of the litter, birth location of the sows (group vs crate), previous farrowing location (group vs crate), and sow parity on total born, live born, number of piglets nursed and weaned, and piglet mortality. Overall pre-weaning mortality of piglets was 27%. Piglet mortality increased with increasing sow parity (from Parity 0 = 22.5% to Parity 3~5 = 34.0%; SE = 3.87%, P = 0.06), which was coincident with increasing litter size (from Parity 0 = 11.1 to Parity 3~5 = 13.5 total piglets born/litter; SE = 0.61, P = 0.01). Piglet mortality was greater for Landrace-sired sows compared with Yorkshire-sired sows (30.8 vs 23.4 ±2.37 %; SE = 3.15%, P = 0.05), possibly due to the larger litter size of Landrace-sired sows (P = 0.05). There was a positive correlation (R2 = 0.75, P < 0.01) between litter size and average pre-weaning mortality of piglets. Sire breed of the litter, sow birth place, and previous farrowing location did not affect piglet mortality. In the current study litter size at farrowing appeared to be the primary factor influencing piglet mortality in a bedded, group farrowing system. 

Evaluation of regional differences in nutrient composition and physical characteristics among six U.S. soybean meal sources

P.E. Urriola¹, M.H. Whitney², N.S. Muley¹, and G.S. Shurson¹

A total of 86 soybean meal (SBM) samples were collected monthly from July 2003 to December 2004 from 6 soybean processing plants (SPP) located in two regions (R): North Carolina (n = 13), South Carolina (n = 13), and Alabama (n = 12) in the southern region (SR), and two Minnesota (n = 14, respectively) and one South Dakota SPP (n = 20) in the Northern region (NR). Subsamples were sent to Iowa Testing Laboratory (Eagle Grove, IA) for proximate and mineral analysis, and to the University of Missouri for amino acid analysis. Additional sub-samples were analyzed for bulk density and particle size. All nutrient values were expressed on a DM (%) basis. Total content of CP (55.23 ± 0.14) and ash (7.02 ± 0.05) content were influenced by year, R, and SPP (P < 0.01). However, crude fat (1.68 ± 0.05) was affected only by SPP (P < 0.01), while crude fiber (3.77 ± 0.03) was affected only by year (P = 0.01). Samples from SR were higher (P = 0.05) in lys (3.45 ± 0.02), met (0.77 ± 0.01), thr (2.08 ± 0.01), and trp (0.76 ± 0.01) than NR (3.38 ±0.01, 0.74 ± 0.01, 2.00 ± 0.01, and 0.73 ± 0.01, respectively). Total AA content and total non-essential AA were higher (P < 0.01) for SR compared to NR. Total CP in SBM was poorly correlated with total lysine content (r2 = 0.39, P < 0.01). Concentrations of P, K and Mn were higher (P < 0.01) in samples from SR than NR. However, Ca, Mg, Na, S and Zn content was similar (P = 0.35) between R. Average particle size (µm) was higher (P > 0.01) in NR (851 ± 33) compared to SR (731 ± 31). Bulk density (kg/m3) was similar (P = 0.41) between R (532 ± 3). Mean particle size and bulk density values were different (P > 0.01) between SPP. Although there were significant differences in nutrient content and physical characteristics of soybean meal samples among regions, year, and SPP, these differences were small and of minimal consequence when formulating practical animal diets.

¹University of Minnesota, St Paul
²University of Minnesota Extension Regional Center, Mankato