In Vitro Maturation and Development of Porcine Oocytes Cultured in a Straw or Dish Using a Portable Incubator with a CO2 Chamber

We investigated the effects of a portable incubator with a CO₂ chamber on the viability and development of porcine oocytes/embryos for their transportation and examined the operational suitability of a straw or dish as a container for culturing the oocytes or embryos in the portable incubator. In the first experiment, the cumulus‐oocyte complexes (COCs) were placed either in a dish or straw; and they were then cultured for 44 h in a standard CO₂ incubator, in the CO₂ chamber in an incubator, or in the CO₂ chamber in a portable incubator. The matured oocytes were fertilized with frozen‐thawed spermatozoa and then cultured in a dish in the standard CO₂ incubator for 8 days. There were no differences in the proportions of oocytes reaching metaphase II stage among the groups. However, the proportions of cleavage and development to blastocysts derived from oocytes matured in a straw were lower than those from oocytes matured in a dish, irrespective of the type of incubator used. In the second experiment, the COCs were matured in a dish in the standard CO₂ incubator, and the matured oocytes were fertilized and then placed either in a dish or straw. These were then cultured for 8 days in the standard CO₂ incubator or portable incubator. Some zygotes cultured in the portable incubator developed to the blastocyst stage. The proportions of cleavage and development to blastocysts were significantly lower for putative zygotes cultured in straw than for those cultured in dish, irrespective of the type of incubator used. Our results indicate that a portable incubator with a CO₂ chamber can maintain the viability and development of oocytes/embryos, but the straw is not a suitable system for in vitro culture of the oocytes/embryos during transportation.     

Factors affecting methane production and mitigation in ruminants

Methane (CH₄) is the second most important greenhouse gas (GHG) and that emitted from enteric fermentation in livestock is the single largest source of emissions in Japan. Many factors influence ruminant CH₄ production, including level of intake, type and quality of feeds and environmental temperature. The objectives of this review are to identify the factors affecting CH₄ production in ruminants, to examine technologies for the mitigation of CH₄ emissions from ruminants, and to identify areas requiring further research. The following equation for CH₄ prediction was formulated using only dry matter intake (DMI) and has been adopted in Japan to estimate emissions from ruminant livestock for the National GHG Inventory Report: Y = −17.766 + 42.793X − 0.849X², where Y is CH₄ production (L/day) and X is DMI (kg/day). Technologies for the mitigation of CH₄ emissions from ruminants include increasing productivity by improving nutritional management, the manipulation of ruminal fermentation by changing feed composition, the addition of CH₄ inhibitors, and defaunation. Considering the importance of ruminant livestock, it is essential to establish economically feasible ways of reducing ruminant CH₄ production while improving productivity; it is therefore critical to conduct a full system analysis to select the best combination of approaches or new technologies to be applied under long-term field conditions.     

Expression levels of taste‐related genes in palate and tongue tip,and involvement of transient receptor potential subfamily M member 5 (TRPM5) in taste sense in chickens

The elucidation of the mechanisms underlying the taste sense of chickens will contribute to improvements in poultry feeding, because the molecular mechanism of chickens’ taste sense defines the feeding behavior of chickens. Here we focused on the gene expressions in two different oral tissues of chickens – the palate, which contains many taste buds, and the tongue tip, which contains few taste buds. Using the quantitative real‐time polymerase chain reaction method, we found that the molecular markers for taste buds of chickens, that is α‐gustducin and vimentin, were expressed significantly highly in the palate compared to the tongue tip. Our analyses also revealed that transient receptor potential subfamily M member 5 (TRPM5), a cation channel involved in taste transduction in mammals, was also highly expressed in the palate compared to the tongue tip. Our findings demonstrated that the expression patterns of these genes were significantly correlated. We showed that the aversion to bitter solution was alleviated by a TRPM5 inhibitor in behavior of chickens. Taken together, our findings enabled us to develop a simple method for screening taste‐related genes in chickens. The use of this method demonstrated that TRPM5 was involved in chickens’ taste transduction, and that a TRPM5 inhibitor can alleviate chickens’ bitter taste perception of feed ingredients.     

Prediction of enteric methane emission from beef cattle in Southeast Asia

We conducted a meta‐data analysis to develop prediction equations to estimate enteric methane (CH₄) emission from beef cattle in Southeast Asia. The dataset was obtained from 25 studies, which included 332 individual observations on nutrient intakes, digestibilities, and CH₄ emissions. Cattle were provided tropical forage or rice straw, with or without concentrates in individual pens equipped with indirect open‐circuit head hood apparatus. The simplest and best equation to predict daily CH₄ emission was CH₄ (g/day) = 22.71 (±1.008) × dry matter intake (DMI, kg/day) + 8.91 (±10.896) [R² = 0.77; root mean square error (RMSE) = 19.363 g/day]. The best equation to predict CH₄ energy as a proportion of gross energy intake (CH₄‐E/GEI, J/100 J) was obtained using DMI per body weight (DMIBW, kg/100 kg), content (g/100 g DM) of ether extract (EE) and crude protein (CP), and DM digestibility (DMD, g/100 g); CH₄‐E/GEI = ?0.782 (±0.2526) DMIBW ? 0.436 (±0.0548) EE ? 0.073 (±0.0218) CP + 0.049 (±0.0097) DMD + 8.654 (±0.6517) (R² = 0.39; RMSE = 1.3479 J/100 J GEI). It was indicated that CH₄ emissions from beef cattle in Southeast Asia are predictable using present developed models including simple indices.     

Effects of voltage strength during electroporation on the development and quality of in vitro‐produced porcine embryos

This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro‐produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm–40 V/mm with five 1‐msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single‐guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.     

Prediction of enteric methane emissions from lactating cows using methane to carbon dioxide ratio in the breath

The aim of this study was to develop prediction equations for methane (CH₄) emissions from lactating cows using the CH₄/carbon dioxide (CO₂) ratio in the breath measured in the automatic milking system (AMS) and to evaluate the predicted values and factors affecting the CH₄/CO₂ ratio. The model development was conducted using a dataset determined in respiration chambers or head boxes (n = 121). Then, gas measurements in the AMS as well as in the head box were carried out with six lactating cows fed one of three different levels of neutral detergent fiber (NDF) content, following a 3 × 3 Latin square experimental design. The obtained equation that is suitable for practical use on farms to predict CH₄ was CH₄ (L/day) = −507 + 0.536 live weight (kg) + 8.76 energy-corrected milk (kg/day) + 5,029 CH₄/CO₂ (adjusted R² = 0.83; root mean square error = 40.8 L/day). Results showed that the predicted values correlated positively with the observed values, the determined CH₄/CO₂ ratio increased with increasing dietary NDF content, and the detected eructation rate was in the normal range. On the other hand, the CH₄/CO₂ ratio was affected by the time interval between measurement and last eating before the measurement.     

Cluster analysis to evaluate disease risk in periparturient dairy cattle

Predicting periparturient disease risk is of immense value to the dairy industry. Periparturient diseases are interrelated with each other; however, predicting the onset risk of these diseases has predominantly been based on a single blood parameter for a single disease. This study examined a new diagnostic method to predict the risk of periparturient diseases. We conducted cluster analysis of multiple blood constituents from 20 Holstein cattle at 1 week post‐partum, and the cattle were divided into two groups, A or B. We then compared the periparturient and early‐lactation blood constituents of these groups. Group B had significantly higher 3‐hydroxybutyric acid concentrations and were suspected to have subclinical ketosis. Group B also had significantly lower calcium concentrations, with a tendency for subclinical hypocalcemia. We also performed discriminant analysis using blood parameters at 1 week post‐partum, which grouped the population into the same two groups as the cluster analysis based on three variables: inorganic phosphorus, calcium, and either phospholipids or total cholesterol. We further showed that these discriminant functions could be used to predict the risk of periparturient disease even before parturition. Our results indicate that cluster analysis with multiple blood constituents is useful for predicting periparturient disease risks.     

Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos^® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.     

Expression of mRNA for sodium-glucose transporter 1 and fatty acid translocase in the ruminant gastrointestinal tract before and after weaning

The mRNA expression of sodium‐glucose transporter 1 (SGLT1) and fatty acid translocase (CD36) in the gastrointestinal tract of Holstein cattle and Saanen goats before and after weaning was investigated. Before weaning, the expression of both SGLT1 and CD36 was highest in the jejunum, relative to the other parts of the gastrointestinal tract in both species. The expression of SGLT1 and CD36 in the duodenum was second highest in the goats. After weaning, SGLT1 and CD36 expression in the small intestine significantly decreased in both species. The expression of both types of transporters was also detected in the forestomach. From these results, it was concluded that the jejunum is probably the major absorption site for glucose and long‐chain fatty acids before weaning, and that the expression of both types of transporters decreases after weaning in cattle and goats.