Removal of sperm tail using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and rats

We examined various methods to enhance the accessibility of intracytoplasmic sperm injection (ICSI) technology to more users by making the technique easier, more efficient, and practical. First, the methods for artificially removing the mouse sperm tail were evaluated. Trypsin treatment was found to efficiently remove the sperm tails. The resultant sperm cells had a lower oocyte activation capacity; however, the use of activated oocytes resulted in the same fecundity as that of fresh, untreated sperm. Pre-activated oocytes were more resistant to physical damage, showed higher survival rates, and required less time per injection. Testing this method in rats yielded similar results, although the oocyte activation method was different. Remarkably, this method resulted in higher birth rates of rat progeny than with conventional methods of rat ICSI. Our method thereby streamlines mouse and rat ICSI, making it more accessible to laboratories across many disciplines.     

An estuarine neritid gastropod, Clithon corona, a potential reservoir of thermostable direct hemolysin-producing Vibrio parahaemolyticus

An estuarine neritid gastropod, Clithon corona, maintained in UV-irradiated recirculating artificial seawater with a salinity of 15 per mil (%o) was found to retain thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus in the gut at significantly higher levels than TDH-non-producing one for at least 14 days. Another estuarine neritid gastropod, C. sowerbianus, was not able to support the preferential survival of TDH-producing organisms. This evidence suggests that, if TDH-producing vibrios are brought to estuaries inhabited by C. corona, repeated ingestion of V. parahaemolyticus by this gastropod could lead to accumulation of TDH-producing vibrios in the estuaries.     

Association of rumen fill score and energy status during the close‐up dry period with conception at first artificial insemination in dairy cows

Recent studies have shown significant associations between prepartum energy status and postpartum fertility in dairy cows; therefore, the assessment of energy status by blood metabolites and metabolic hormones and suitable improvement of management during the prepartum period may enhance reproductive performance. Rumen fill score (RFS) is associated with feed intake; however, it is unknown whether RFS is also related to blood parameters. Therefore, this study investigated the relationship between RFS and energy status during the prepartum period, and their associations with conception at first artificial insemination (AI) after parturition. In 42 multiparous Holstein cows, RFS assessment and blood sampling were carried out twice a week during 3 weeks of the peripartum period. Ovarian cycles until AI were evaluated by measuring milk progesterone levels. Before calving, positive correlations were observed between RFS and total cholesterol, and RFS did not change in pregnant cows at first AI after parturition, whereas in non‐pregnant cows, RFS decreased gradually as the calving day approached. After calving, non‐pregnant cows showed lower energy status compared with pregnant cows, and some non‐pregnant cows showed anovulation and cessation of estrous cycle. In conclusion, RFS during the close‐up dry period is related to real‐time energy status, and is associated with postpartum energy status and conception at first AI in dairy cows. © 2016 Japanese Society of Animal Science     

Comparison of 3 different reinforcements of learning in dogs (Canis familiaris)

Effective dog training involves reinforcement of the dog's correct actions in response to specific cues given by the trainer. Food is usually selected as the primary reinforcer (reward), although this selection does not necessarily account for the learning preferences of individual dogs. We evaluated the relationship between reward and learning efficiency. Fifteen dogs were allocated to 3 different reward groups (food, stroking, and praise) and trained by an identical process. The food reward was the only one that shortened the time taken for the response to the command to be completed. However, this difference occurred only in the early training stages and not later in the training process.     

Inosine‐5'‐monophosphate is a candidate agent to resolve rigor mortis of skeletal muscle

The object of the present study was to reveal the action of inosine‐5'‐monophosphate (IMP) toward myofibrils in postmortem muscles. IMP solubilized isolated actomyosin within a narrow range of KCl concentration, 0.19‐0.20 mol/L, because of the dissociation of actomyosin into actin and myosin, but it did not solubilize the proteins in myofibrils with 0.2 mol/L KCl. However, IMP could solubilize both proteins in myofibrils with 0.2 mol/L KCl in the presence of 1 m mol/L pyrophosphate or 1.0–3.3 m mol/L adenosine‐5'‐diphosphate (ADP). Thus, we presumed that pyrophosphate and ADP released thin filaments composed of actin, and thick filaments composed of myosin from restraints of myofibrils, and then both filaments were solubilized through the IMP‐induced dissociation of actomyosin. Thus, we concluded that IMP is a candidate agent to resolve rigor mortis because of its ability to break the association between thick and thin filaments.     

Renal Tubular Cyst Formation in Newborn Rats Treated with p-Cumylphenol

In this study, we investigated the sequential changes in the development of renal tubular cysts in newborn rats treated with p-cumylphenol (PCP). Fifteen animals per sex were treated orally with 300 mg/kg/day of PCP for up to 18 days from postnatal day (PND) 4 and were sacrificed on PNDs 8, 12, 19 and 22 and after a 7 day recovery period. On PNDs 8 and 12, slight dilatation of the collecting ducts was frequently observed in the medulla and slight papillary necrosis was also noted in some cases. These dilated collecting ducts were lined with slightly hyperplastic epithelial cells. On PNDs 19 and 22, multiple large cystic changes arising from the collecting ducts in the outer medulla were seen. These cystically dilated ducts were also lined with hyperplastic epithelial cells. During the dosing period, the labeling index of proliferating cell nuclear antigen in the collecting duct epithelium was higher in the PCP-treated group than in the control group at all time points. After a 7 day recovery period, the cystic change still remained, although the cell density was decreased and the epithelial cells became flattened. On the other hand, basophilic tubules with peritubular lymphoid cell infiltration were multifocally observed in the cortex. In conclusion, PCP induced multiple renal cysts that developed in the collecting ducts of the outer medulla in neonatal rats, and it is suggested that epithelial cell proliferation may play some roles in the induction of cystic lesions.     

Comparison of enzyme activities in plasma and leukocytes in dairy and beef cattle

Concentrations of plasma glucose, immunoreactive insulin (IRI) and free fatty acid (FFA) and activities of enzymes related to energy metabolism and lactate dehydrogenase (LDH) isoenzyme pattern in plasma and leukocytes were investigated in lactating Holstein cows (dairy cattle) and fattening Japanese Black Wagyu x Holstein steers (beef cattle). IRI concentrations and LDH and malate dehydrogenase (MDH) activities in the plasma of beef cattle were significantly higher than those in dairy cattle. The cytosolic ratio of MDH/LDH activity in the leukocytes of beef cattle was significantly higher than that of dairy cattle. These findings might be associated with the different energy metabolism between dairy and beef cattle.     

Chemical characterization of milk oligosaccharides of the common wombat (Vombatus ursinus)

Previous structural characterizations of marsupial milk oligosaccharides have been performed in the tammar wallaby, red kangaroo, koala, common brushtail possum and the eastern quoll. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of wombat milk carbohydrate were characterized in this study. Neutral and acidic oligosaccharides were isolated from the carbohydrate fractions of two samples of milk of the common wombat and characterized by ¹H‐nuclear magnetic resonance spectroscopy. The structures of six neutral saccharides were found to be Gal(β1‐4)Glc (lactose), Gal(β1‐3)Gal(β1‐4)Glc (3'‐galactosyllactose), Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc (3',3”‐digalactosyllactose), Gal(β1‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc, Gal(β1‐3)Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (galactosyl lacto‐N‐novopentaose I) and Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (lacto‐N‐novooctaose), while those of six acidic saccharides were Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐4)Glc. (sialyl 3'‐galactosyllactose), Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc (sialyl 3',3”‐digalactosyllactose), Neu5Ac(α2‐3)Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (sialyl lacto‐N‐novopentaose a), Gal(β1‐3)[Neu5Ac(α2‐3)Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (sialyl lacto‐N‐novopentaose c), Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc,, Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc and Gal(β1‐3)Gal(β1‐3)[Neu5Ac(α2‐3)Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc. In addition, small amounts of sulfated oligosaccharides but no oligosaccharides containing Neu5Gc or α(2–6) linked Neu5Ac were detected.     

Extracellular cyclophilin A possesses chemotaxic activity in cattle

Cyclophilin A (CyPA) was originally discovered in bovine thymocytes as a cytosolic binding protein of the immunosuppressive drug cyclosporine A. Recent studies have revealed that in mice and humans, CyPA is secreted from cells in injured or infected tissues and plays a role in recruiting inflammatory cells in those tissues. Here we found that in cattle abundant level of extracellular CyPA was observed in tissues with inflammation. To aid in investigating the role of extracellular CyPA in cattle, we generated recombinant bovine CyPA (rbCyPA) and tested its biological activity as an inflammatory mediator. When bovine peripheral blood cells were treated with rbCyPA in vitro, we observed that rbCyPA reacts with the membranous surface of granulocytes, monocytes and lymphocytes. Chemotaxis analysis showed that the granulocytes migrate toward rbCyPA and the migration is inhibited by pre-treatment with an anti-bovine CyPA antibody. These results indicate that, as for mice and humans, extracellular CyPA possesses chemotactic activity to recruit inflammatory cells (e.g., granulocytes) in cattle, and could thus be a potential therapeutic target for the treatment of inflammation.     

Strawberry cultivar identification based on hypervariable SSR markers

We genotyped strawberry cultivars by two newly selected and two previously reported SSR markers. All four markers produced interpretable electropherograms from 75 accessions consisting of 72 Fragaria × ananassa cultivars or lines and three octoploid Fragaria species accessions. These SSR markers were highly polymorphic; in particular, one of the newly developed markers, FxaHGA02P13, was capable of distinguishing all of the accessions except for a mutant strain that was derived from another accession in the set. When two markers were combined, all 48 full-sib individuals could be distinguished. Fingerprinting patterns were reproducible between multiple samples, including the leaves, sepals, and fruit flesh of the same accession. Principal-coordinate analysis of the 75 accessions detected several groups, which reflect taxon and breeding site. Together with other available markers, these SSR markers will contribute to the management of strawberry genetic resources and the protection of breeders’ rights.