Selected Aspects of Advanced Porcine Reproductive Technology

In vitro fertilization (IVF) of in vitro matured (IVM) oocytes in pigs has become the most popular method of studying gametogenesis and embryogenesis in this species. Furthermore, because of recent advances in in vitro culture (IVC) of IVM–IVF embryos, in vitro production (IVP) of embryos now enables us to generate viable embryos as successfully as for in vivo -derived embryos and with less cost and in less time. These technologies contribute not only to developments in reproductive physiology and agriculture but also to the conservation of porcine genetic resources and the production of cloned or genetically modified pigs. However, in IVP, there still remains the problem of abnormal ploidy, which is caused by performing procedures under non-physiological conditions. In recent years, unique technologies such as intracytoplasmic sperm injection (ICSI) or xenografting of gonadal tissue into immunodeficient experimental animals have been developed to help conserve gamete resources. These technologies combined with IVP are expected to be useful for the conservation of gametes from important genetic resources. Here, we discuss the developmental ability and normality of porcine IVP embryos and also the utilization of ICSI and xenografting in advancing biotechnology in pigs.     

Embryo collection from pigs post‐pseudopregnancy induced by estradiol dipropionate

We aimed to define whether embryo collection carried out after pseudopregnancy was of similar outcome and quality as after artificial abortion. To induce pseudopregnancy, 30 gilts or sows were given 20 mg intramuscular estradiol dipropionate (EDP) 10–11 days after the onset of estrus. Ten additional pigs were inseminated artificially at natural estrus as a control group. Prostaglandin F_2α (PGF_2α) was administered twice with a 24 hr interval beginning 15, 20, or 25 days after EDP‐treatment (n = 10 per group) or between 23 and 39 days after artificial insemination in control pigs. Following this, all pigs were given 1,000 IU equine chorionic gonadotropin and 500 IU human chorionic gonadotropin (hCG) and then inseminated. Embryos were recovered 6 or 7 days after hCG treatment and outcome was recorded. There was no significant difference in the number of normal embryos collected from the pigs with PGF_2α initiated at different time points or from the control group. Embryonic developmental stages 7 days after hCG treatment also did not differ among groups. These results indicate that the use of EDP to induce pseudopregnancy, followed by PGF_2α administration to synchronize estrus for subsequent embryo harvest, is a suitable alternative to the artificial abortion method.     

Sucrose assists selection of high‐quality oocytes in pigs

We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes.     

Oncolytic reovirus therapy: Pilot study in dogs with spontaneously occurring tumours

Oncolytic virotherapy is a novel treatment involving replication‐competent virus in the elimination of cancer. We have previously reported the oncolytic effects of reovirus in various canine cancer cell lines. This study aims to establish the safety profile of reovirus in dogs with spontaneously occurring tumours and to determine a recommended dosing regimen. Nineteen dogs with various tumours, mostly of advanced stages, were treated with reovirus, ranging from 1.0 × 10⁸ to 5.0 × 10⁹ TCID₅₀ given as intratumour injection (IT) or intravenous infusion (IV) daily for up to 5 consecutive days in 1 or multiple treatment cycles. Adverse events (AEs) were graded according to the Veterinary Cooperative Oncology Group‐ Common Terminology Criteria for Adverse Events (VCOG‐CTCAE) v1.1 guidelines. Viral shedding, neutralizing anti‐reovirus antibody (NARA) production and immunohistochemical (IHC) detection of reovirus protein in the tumours were also assessed. AE was not observed in most dogs and events were limited to Grade I or II fever, vomiting, diarrhoea and inflammation of the injected tumour. No infectious virus was shed and all dogs had elevated NARA levels post‐treatment. Although IHC results were only available in 6 dogs, 4 were detected positive for reovirus protein. In conclusion, reovirus is well‐tolerated and can be given safely to tumour‐bearing dogs according to the dosing regimen used in this study without significant concerns of viral shedding. Reovirus is also potentially effective in various types of canine tumours.     

Mechanical and ecophysiological significance of the form of a young Acer rufinerve tree: vertical gradient in branch mechanical properties

Most tree biomechanics models assume uniformity of mechanical properties within a tree, and only a few studies have focused on differences in mechanical status among branches. We examined mechanical properties of 49 branches of two 10-year-old trees of Acer rufinerve Sieb. et Zucc. For each branch, bending moment due to its own fresh mass, elastic modulus, section modulus and flexural stiffness were obtained. Elastic modulus of the branch was correlated with the density and thickness of the fiber cell wall and decreased with crown depth, indicating that branches at lower positions were more elastic than branches at upper positions. Compared to lower branches, upper branches were less inclined, possessed thicker growth rings, more long shoots and were subject to smaller stresses. The leaf arrangement in the upper branches might be effective in transmitting more light to the lower branches. In contrast, the lower branches were more inclined toward the horizontal and subject to greater stresses than the upper branches. Lower branch inclinations were attributed to smaller dry matter investment in diameter growth. Upper and lower branch inclinations were slightly greater and smaller, respectively, than those predicted by beam theory. The alleviation in inclination of the lower branches is probably caused by negative gravitropic responses such as tension wood formation or upward shoot elongation, or both. The horizontal display of leaves in the lower branches would be effective in light interception. The reduction in cost of the lower branches can be adaptive because they have a shorter life expectancy than the upper branches. The results showed that an adaptive tree form is realized by a vertical gradient in branch mechanical properties.     

Estimation of stiffness and strength in timber knee joints with adhesive and verification by experiment

The use of adhesive joints is gradually increasing, especially those with glued-in steel rods (GIRs). There are, however, some problems with the design methods when used for moment-transmitting applications. In this article, design methods for GIRs and cross-lapped glued joints (CLJs) are proposed. For CLJs, we made a hypothesis that both rotational deformation of CLJ and stress of the glue line occurred with bending and shearing deformation of the timber. Using this hypothesis and Kelvin’s theorem, a mechanical model of CLJ is proposed. For GIRs, the axial stress component of the rod and the lateral stress component of the rod were taken into account using the theory of a beam on an elastic foundation. From the comparisons between calculations and experimental results, it was recognized that the stiffness and strength of CLJs and GIRs could be predicted precisely using our proposed models.     

Effects of polyethylene glycol and a synthetic ice blocker during vitrification of immature porcine oocytes on survival and subsequent embryo development

We evaluated the effects of polyethylene glycol (PEG) and Supercool X‐1000 (SC) as supplements during the vitrification of immature cumulus‐enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG‐, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG‐ groups; however, all values were lower than those in the non‐vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC‐, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC‐ groups but lower than those in the non‐vitrified control. The percentage of cleavage in the SC‐ group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non‐vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts.     

Nuclear Replacement of In Vitro‐Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method

The objective of the present study was to establish a method for nuclear replacement in metaphase‐II (M‐II) stage porcine oocytes. Karyoplasts containing M‐II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro‐matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona‐free M‐II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2–77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2–32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0–15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri‐Fusion) is an effective method for producing M‐II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.     

Effects on Maize Starch Properties of Heat-treatment with Water–ethanol Mixtures

Maize starch (1 part) was heated at 120 °C for 20 min in water–ethanol mixtures (5 volumes), in which the water content varied over the range 0–70%, and the product solvent-exchanged with acetone. On treatment with mixtures containing 40–60% water, the swelling power and solubility at 97 °C decreased and the differences between these values measured at 25 and 97 °C narrowed. The properties of starch treated with a mixture containing 30% water resembled those of heat–moisture treated starch. The values of ΔH and ΔS for swelling were greatly decreased by treatment in mixtures with a water content greater than 20–40% (0·39–0·63 mole fraction). Furthermore, the digestibility ratio ofalpha-amylase (endo-type) to glucoamylase (exo-type) was at a maximum after treatment with a mixture containing 30% water. These findings suggest that rearrangements between the amylose and amylopectin in starch granules are occurring after treatment with mixtures containing more than 30% water. These changes in the starch granule are enhanced when the treatment temperature is increased to 100–130 °C.     

Evaluation of bone toxicity in various bones of aged rats

The aim of the present study was to provide a method for evaluating bone toxicity induced by drugs in various bones in aged rats. Male Crl:CD (SD) rats at 46 weeks of age were administered 15 mg/m(2) body surface area of doxorubicin, which effects the growth plate in weanling rats, weekly for 9 weeks by intravenous injection, and the femur, sternum, humerus and tibia were examined histopathologically. In the doxorubicin-treated group, thinning of the growth plate was remarkably observed in the proximal tibia and humerus; however, these changes were not observed in other regions. In addition, the osteoclast number per bone perimeter in the proximal tibia was significantly higher than others in control aged rat. Thus, recognizing the various histological reactions related to the time of epiphyseal closure is important for evaluating bone toxicity in aged rats.