A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.     

Investigations on the influence of duodenal histidine infusion on nitrogen and amino acid turnover of growing German holstein bulls

The effect of a continuous duodenal infusion of L‐histidine (His) (8 g/d) on the retention of nitrogen was investigated in two experiments (I, II), each of which was carried out using two young bulls. In Exps. I and II, the animals (150–250 kg BW) were fitted with a re‐entrant cannula in the proximal duodenum and were fed diets containing 125 g CP/kg DM and 11.5 MJ ME/kg DM. A third experiment (III) using two young bulls (140–200 kg BW) fitted with a simple T‐cannula was carried out infusing 6 g L‐His. The animals were fed a low protein diet (94 g CP/kg DM and 11 MJ ME/kg DM). The study was done to find out whether or not L‐His is the first limiting amino acid (AA) for growing ruminants.

N retention was 28 and 31, 38 and 38, 22 and 24 g/d without L‐His infusion and with L‐His infusion for Exps. I, II and III, respectively. Both in the experiments with a standard protein supply (I, II) and in the experiment with reduced protein supply (III), no significant differences were found between periods with and without infusion of L‐His. The utilisation of duodenal NAN varied between 39% and 50% and was also not significantly influenced by the duodenal infusion of L‐His. No significant effect was observed on the flow of AA into the duodenum. The faecal excretion of AA was also not significantly influenced by the infusion of L‐His. The utilisation of individual amino acids as calculated by the ratio of retained AA to intestinal apparently digested AA, did not differ significantly following the duodenal infusion of L‐His. As expected, the utilisation of His decreased. Of the different essential AA, L‐His was the most utilised (80%) followed by Arg (72%), Met (60%), Leu (45%) and Lys (44%), during periods without supplementation of L‐His.

It is concluded that the intestinal supply of L‐His from the basal diet was sufficient for the potential growth level of animals under these experimental conditions. In all AA present at the proximal duodenum, L‐His could have at first a limiting effect on the performance of growing young bulls with high body gain. Arg and Met, but not Lys, could be second or co‐limiting AA.     

Consequences of a low litter birth weight phenotype for postnatal lean growth performance and neonatal testicular morphology in the pig

The consequences of a low litter average birth weight phenotype for postnatal growth performance and carcass quality of all progeny, and testicular development in male offspring, were investigated. Using data from 25 sows with one, and 223 sows with two consecutive farrowing events, individual birth weight (BW) was measured and each litter between 9 and 16 total pigs born was classified as low (LBW), medium (MBW) or high (HBW) birth weight: low and high BW being defined as >1 standard deviation below or above, respectively, the population mean for each litter size. Litter average BW was repeatable within sows. At castration, testicular tissue was collected from 40 male pigs in LBW and HBW litters with individual BW close to their litter average BW and used for histomorphometric analysis. LBW piglets had a lower absolute number of germ cells, Sertoli cells and Leydig cells in their testes and a higher brain : testis weight ratio than HBW piglets. Overall, LBW litters had lower placental weight and higher brain : liver, brain : intestine and brain : Semitendinosus muscle weight ratios than MBW and HBW litters. In the nursery and grow–finish (GF) phase, pigs were kept in pens by BW classification (9 HBW, 17 MBW and 10 LBW pens) with 13 males and 13 females per pen. Average daily gain tended to be lower in LBW than HBW litters in lactation (P = 0.06) and throughout the nursery and GF phases (P < 0.01), resulting in an increasing difference in body weight between LBW, MBW and HBW litters (P < 0.05). Average daily feed intake was lower (P < 0.001) in LBW than HBW litters in the nursery and GF phases. Feed utilization efficiency (feed/gain) was similar for LBW and HBW litters in the nursery, but was lower (P < 0.001) in HBW than LBW litters in the GF phase. By design, slaughter weight was similar between BW classifications; however, LBW litters needed 9 more days to reach the same slaughter weight than HBW litters (P < 0.001). BW classification did not affect carcass composition traits. In conclusion, LBW litters showed benchmarks of intrauterine growth retardation, LBW had a negative impact on testicular development and germ and somatic cell populations, and was associated with decreased postnatal growth during all phases of production; however, no measurable effect on carcass composition traits was established.     

Can plant litter affect net primary production of a typical steppe in Inner Mongolia?

Question: Litter (dead leaves or stems) affects production by conserving soil moisture. However, that role is not clear for grasslands where most precipitation falls during the growing season when the demand for water is high. Our question was: Does litter affect forage production in such an environment? Location: Typical steppe, Inner Mongolia. Methods: We examined the role of plant litter in two experiments where litter was either removed or added in a protected or heavily grazed site, respectively, in autumn and in spring in a split plot design. The treatments (control, moderate and heavy litter application) were applied once in five replications but repeated at new locations in each of 3 years. This was done to examine only the direct effect of litter on annual net primary production and selected plant characteristics and not potential secondary effects. We also measured soil moisture and soil temperature. Results: Removing litter caused a reduction in the amount of grass (Leymus chinensis) that was produced, but litter addition caused an inconsistent effect among years, with moderate applications producing the most positive effects. Litter removal resulted in shorter and less dense plants of L. chinensis and Carex duriuscula, while heavy litter addition in autumn reduced plant height of both Cleistogenes squarrosa and C. duriuscula. Conclusions: Litter was effective for enhancing soil moisture status and reducing soil heat units in the typical steppe of Inner Mongolia. Therefore, litter mass may serve as an index of grassland health in such environments.     

Assessing the utility of metabarcoding for diet analyses of the omnivorous wild pig (Sus scrofa)

Wild pigs (Sus scrofa) are an invasive species descended from both domestic swine and Eurasian wild boar that was introduced to North America during the early 1500s. Wild pigs have since become the most abundant free‐ranging exotic ungulate in the United States. Large and ever‐increasing populations of wild pigs negatively impact agriculture, sport hunting, and native ecosystems with costs estimated to exceed $1.5 billion/year within the United States. Wild pigs are recognized as generalist feeders, able to exploit a broad array of locally available food resources, yet their feeding behaviors remain poorly understood as partially digested material is often unidentifiable through traditional stomach content analyses. To overcome the limitation of stomach content analyses, we developed a DNA sequencing‐based protocol to describe the plant and animal diet composition of wild pigs. Additionally, we developed and evaluated blocking primers to reduce the amplification and sequencing of host DNA, thus providing greater returns of sequences from diet items. We demonstrate that the use of blocking primers produces significantly more sequencing reads per sample from diet items, which increases the robustness of ascertaining animal diet composition with molecular tools. Further, we show that the overall plant and animal diet composition is significantly different between the three areas sampled, demonstrating this approach is suitable for describing differences in diet composition among the locations.     

Why are most EU pigs tail docked? Economic and ethical analysis of four pig housing and management scenarios in the light of EU legislation and animal welfare outcomes

To limit tail biting incidence, most pig producers in Europe tail dock their piglets. This is despite EU Council Directive 2008/120/EC banning routine tail docking and allowing it only as a last resort. The paper aims to understand what it takes to fulfil the intentions of the Directive by examining economic results of four management and housing scenarios, and by discussing their consequences for animal welfare in the light of legal and ethical considerations. The four scenarios compared are: ‘Standard Docked’, a conventional housing scenario with tail docking meeting the recommendations for Danish production (0.7 m²/pig); ‘Standard Undocked’, which is the same as ‘Standard Docked’ but with no tail docking, ‘Efficient Undocked’ and ‘Enhanced Undocked’, which have increased solid floor area (0.9 and 1.0 m²/pig, respectively) provision of loose manipulable materials (100 and 200 g/straw per pig per day) and no tail docking. A decision tree model based on data from Danish and Finnish pig production suggests that Standard Docked provides the highest economic gross margin with the least tail biting. Given our assumptions, Enhanced Undocked is the least economic, although Efficient Undocked is better economically and both result in a lower incidence of tail biting than Standard Undocked but higher than Standard Docked. For a pig, being bitten is worse for welfare (repeated pain, risk of infections) than being docked, but to compare welfare consequences at a farm level means considering the number of affected pigs. Because of the high levels of biting in Standard Undocked, it has on average inferior welfare to Standard Docked, whereas the comparison of Standard Docked and Enhanced (or Efficient) Undocked is more difficult. In Enhanced (or Efficient) Undocked, more pigs than in Standard Docked suffer from being tail bitten, whereas all the pigs avoid the acute pain of docking endured by the pigs in Standard Docked. We illustrate and discuss this ethical balance using numbers derived from the above-mentioned data. We discuss our results in the light of the EU Directive and its adoption and enforcement by Member States. Widespread use of tail docking seems to be accepted, mainly because the alternative steps that producers are required to take before resorting to it are not specified in detail. By tail docking, producers are acting in their own best interests. We suggest that for the practice of tail docking to be terminated in a way that benefits animal welfare, changes in the way pigs are housed and managed may first be required.     

Healthy birth weight results in higher vitamin A storage in neonate piglets administered high-dose supplements

A proposed intervention for newborn infants in countries with suspected vitamin A (VA) deficiency is to administer 50,000 IU retinyl palmitate at birth to reduce mortality risk. However, no studies have investigated birth weight effects. In this study, low birth weight (LBW; <1 kg, n = 18) and healthy birth weight (HBW) piglets (>1.5 kg, n = 18) from VA-depleted sows were dosed with 25,000 or 50,000 IU retinyl palmitate (26.2 or 52.4 µmol retinol equivalents) at birth to compare VA reserves. Blood was collected at varying times (n = 3–5/time/dose), and piglets were killed at 12 or 24 h for blood, liver, kidneys, spleen, lungs, adrenal gland, and intestinal contents. HBW piglets had significantly higher birth, death, and organ weights than LBW (P < 0.0001 for all). HBW and LBW piglets, which received VA, had higher liver and kidney VA concentrations (0.18 ± 0.09, 0.24 ± 0.10 µmol/g liver and 13.4 ± 4.1, 14.2 ± 4.5 nmol/g kidney, respectively) than controls (n = 10) (0.051 ± 0.01 µmol/g liver and 1.01 ± 0.43 nmol/g kidney) (P = 0.0061 and < 0.0001, respectively). Total liver (9.75 ± 5.16 µmol) and kidney retinol (204 ± 79.1 nmol) were higher in HBW than LBW piglets (P < 0.0001). Extrahepatic tissues, except lung, had higher VA concentration than controls (P < 0.0001). Serum retinol and ester concentrations were higher in treated than control piglets (P = 0.0028, P < 0.0001, respectively), and significantly changed during the times sampled (P = 0.022, P = 0.011, respectively). Peak serum retinyl ester concentrations, which occurred at 3 h, were higher in piglets that received 50,000 IU (4.2 ± 4.4 µmol/L) than 25,000 IU (2.7 ± 2.3 µmol/L) (P = 0.031). Regardless of dose amount, HBW piglets stored more supplemental VA than LBW piglets when administered at birth.     

猪瘟病毒石门强毒株和兔化弱毒疫苗株E2蛋白糖基化位点差异分析

猪瘟病毒强毒株和兔化弱毒疫苗株E2糖蛋白分别含有5个和6个潜在的糖基化位点,其中986N是兔化弱毒疫苗株所特有的。为了分析二者糖基化位点差异及其影响,将去掉信号肽和跨膜区的猪瘟病毒石门强毒株(Shi-men)和兔化弱毒疫苗株(HCLV)E2基因置于蜂素信号肽序列下游,使其在Sf9细胞内表达重组Shimen-E2和HCLV-E2蛋白。结果显示,重组E2蛋白以二聚体的形式分泌表达于细胞培养液中,但二者分子量存在差异。用endo H和PNGase F对纯化后的重组E2蛋白进行去糖基化处理后,二者分子量大小变成一致,证实石门强毒株和兔化弱毒株E2蛋白分子量大小的差异可能是由于糖基化程度的差异所致。对986N糖基化位点进行定点突变后发现,突变后的Shimen-E2与野生型HCLV-E2分子量大小一致,而突变后的HCLV-E2与野生型Shimen-E2分子量大小一致,表明Shimen-E2和HCLV-E2分子量大小的差异的确是由于986N糖基化位点的差异引起的。     

内江猪IL-18基因的克隆及原核表达

以ConA刺激的内江猪外周血单核淋巴细胞(PBMC)为模板,采用RT-PCR技术扩增出猪IL-18全长基因,克隆其成熟蛋白的编码基因(471bp)至pMD18-T克隆载体,经双酶切和测序获得阳性克隆。通过EcoRⅠ/XhoⅠ双酶切及连接反应,构建了pET-32a( )-IL-18原核表达质粒。经双酶切和DNA测序证实重组质粒构建正确,将阳性重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE,Western-blot证实表达出35kD左右的融合蛋白。经实验确定重组内江猪IL-18蛋白诱导表达的最佳条件为IPTG 0.2mmol/L,30℃诱导培养5h。在最佳表达条件下,pIL-18的相对含量为59.6%。内江猪IL-18的原核表达为重组IL-18的纯化及其生物学功能的进一步研究提供了基础。     

Behavioral states in space and time: understanding landscape use by an invasive mammal

Animal movement models can be used to understand species behavior and assist with implementation of management activities. We explored behavioral states of an invasive wild pig (Sus scrofa) population that recently colonized central Michigan, USA, 2014–2018. To quantify environmental factors related to wild pig movement ecology and spatio-temporal landscape use, we predicted wild pig behavioral states relative to land cover type, landscape structure (i.e., edge and patch cohesion), and weather conditions. We used global positioning system (GPS)-collars and monitored 8 wild pigs from 2014–2018. We fit local convex hulls and calculated movement metrics revealing 3 wild pig behavioral states (resting, exploratory, and relocating) and constructed a 3-level model to predict behavioral state probabilities relative to biotic and abiotic conditions. Probabilities of exploratory and resting behaviors were higher nearer to riparian and open herbaceous cover types (oftentimes emergent marsh), indicating that these cover types provided security cover during activity and bedding. Hard mast cover types had a strong positive association with relocating behaviors. More cohesive patches of agriculture and shrub cover types were associated with higher probabilities of exploratory behaviors, while resting was more likely in continuous patches of agriculture (mostly mid-summer corn). The probability of exploratory behaviors increased exponentially with warming ambient temperature. Our results may be used by managers to develop control strategies conducive to landscape and environmental conditions where the likelihood of encountering wild pigs is highest or targeting wild pigs when in a behavioral state most vulnerable to a particular removal technique.