Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization

Electroporation is the technique of choice to introduce an exogenous gene into embryos for transgenic animal production. Although this technique is practical and effective, embryonic damage caused by electroporation treatment remains a major problem. This study was conducted to evaluate the optimal culture system for electroporation‐treated porcine embryos by supplementation of chlorogenic acid (CGA), a potent antioxidant, during in vitro oocyte maturation. The oocytes were treated with various concentrations of CGA (0, 10, 50, and 100 μmol/L) through the duration of maturation for 44 hr. The treated oocytes were then fertilized, electroporated at 30 V/mm with five 1 msec unipolar pulses, and subsequently cultured in vitro until development into the blastocyst stage. Without electroporation, the treatment with 50 μmol/L CGA had useful effects on the maturation rate of oocytes, the total cell number, and the apoptotic nucleus indices of blastocysts. When the oocytes were electroporated after in vitro fertilization, the treatment with 50 μmol/L CGA supplementation significantly improved the rate of oocytes that developed into blastocysts and reduced the apoptotic nucleus indices (4.7% and 7.6, respectively) compared with those of the untreated group (1.4% and 13.0, respectively). These results suggested that supplementation with 50 μmol/L CGA during maturation improves porcine embryonic development and quality of electroporation‐treated embryos.     

Structural differences between myofibrillar protein,paratropomyosin, and tropomyosin as revealed by high‐performance liquid chromatography

Paratropomyosin (PTM) composes myofibril functions to weaken the rigor linkages formed between actin and myosin during postmortem aging of muscles. PTM has the similar physico‐chemical properties as tropomyosin (TM) that is a regulatory protein of myofibrils. So far, it is unclear whether PTM is definitely different from TM, because the primary structure of PTM has not been determined yet. The aim of this study was to clarify structural difference of PTM from TM. PTM was prepared by column chromatography immediately after slaughter from broiler breast muscle, and purified by high‐performance liquid chromatography (HPLC). Purified PTM was successfully separated from TM, and the recovered PTM molecule was reduced with dithiothreitol to separate again by HPLC. Two subunits were obtained and peptides from each digested subunit by V8 protease were recovered by HPLC, and then amino acid sequences of the peptides were analyzed by protein sequencing. As a result, some amino acid residues were replaced from that of TMα1 isoform which is the major isoform of TM, and also was different between the two subunits. Therefore, it is concluded that PTM clearly differs from TM and it is suggested that functional difference in PTM from TM is attributed to amino acid replacements in subunits composing PTM.     

Effect of increased concentrate allotment before evening grazing on herbage intake,nitrogen utilization and rumen fermentation in dairy cows grazed on perennial ryegrass pasture

Two experiments were conducted to elucidate the effect of increased concentrate allotment before evening grazing on herbage intake, nitrogen utilization and rumen fermentation in dairy cows. In experiment 1, nine lactating cows were grazed in the morning and evening sessions (2.5 h each). The cows were allocated to treatments of three concentrate allotment levels before the evening grazing session by altering proportions to daily total offered; 25%, 50% and 75%. Daily herbage dry matter intake quadratically decreased with increasing the concentrate allotment levels (P < 0.05). Nitrogen utilization was similar among treatments. To investigate diurnal changes in rumen fermentation, a second experiment was conducted where six ruminally cannulated non‐lactating dairy cows grazed in the morning and evening sessions (3 h each) were subjected to the same treatments as experiment 1. Total volatile fatty acid concentration in the rumen linearly increased with increasing the concentrate allotment levels throughout the pre‐evening grazing session to the post‐morning grazing session (P < 0.01). The results indicate that dairy cows reduce daily herbage intake but do not alter nitrogen utilization with increasing concentrate allotment before evening grazing. © 2016 Japanese Society of Animal Science     

Estrous cycle based on blood progesterone profiles and changes in vulvar appearance of Malayan tapirs (Tapirus indicus)

The progesterone (P(4)) profiles and macroscopic vulvar changes of female Malayan tapirs were investigated in order to understand their fundamental reproductive physiology and to search for visual indicators of estrus. Blood was collected once or twice a week from seven female Malayan tapirs kept at four zoos. Serum or plasma P(4) concentrations were determined by radioimmunoassay. The P(4) concentrations changed cyclically throughout the years, and a total of 56 cycles was confirmed in the seven females. The length of the estrous cycle based on the P(4) profiles was 43.6+/-2.0 days; however, this mean includes great variation in length, from 21 to 84 days. Mucous discharge from the vulva and vulvar swelling were seen when the P(4) concentrations were low before the beginning of a rise in most cases. In conclusion, captive female Malayan tapirs have variations of approximately 1 to 3 months in estrous cycle length, and visual changes in the vulva are helpful in estimating estrus in female Malayan tapirs.     

Effects of the presence of grazing‐experienced heifers on the development of foraging behavior at the feeding station scale for first‐grazing season calves

In this study, effects of grazing‐experienced heifer presence on foraging behavior development at the feeding station (FS) scale for first‐grazing season calves were determined. A group of four calves grazing alone (C), and another comprising four calves (Wc) and three grazing‐experienced heifers (Wh), were alternately stocked every day for 2 h on the same pasture for 26 days. The foraging time budget for Wc was significantly longer than that for C (P < 0.05) on day 7, and was similar to that for Wh on all days. Bite rate per FS was significantly higher for Wc (15.5 bite/min) than for C (13.2 bite/min) from day 1 to 14 (33.4 vs. 29.0 bite/min, respectively; P < 0.01) and significantly lower than that for Wh on all days. The number of steps taken between adjacent FSs by Wc (4.7) was significantly lower than that for C (7.2) on days 1 and 14 (2.1 vs. 2.9 steps, respectively; P < 0.01) and was similar to Wh on all days. This suggests that grazing‐experienced heifers have positive effects on the foraging behavior development of new‐season grazing calves through 14 days after the start of stocking.     

Changes in GnRH levels in the brain and pituitary gland during migrations of sockeye salmon and chum salmon

Levels of two moleculer types of gonadotropin-releasing hormone (GnRH), salmon GnRH (sGnRH) and chicken GnRH–II (cGnRH–II) in the various brain regions and pituitary gland of sockeye salmon (Oncorhynchus nerka) and chum salmon (O. keta) during smoltification and spawning migration, respectively, were measured using specific time-resolved fluoroimmunoassay (TR-FIA) systems. Changes in sGnRH levels in different brain regions tended to be specifically synchronized with serum thyroid hormone or pituitary gonadotropin (GTH) levels during smoltification and spawning migration, respectively. In contrast, cGnRH–II levels did not show such synchronized changes. SGnRH and cGnRH–II in various brain regions might have different roles during smoltification and spawning migration of salmonid fishes.     

Otolith microstructure of chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis>: formation of sea entry check and daily deposition of otolith increments in seawater conditions

To apply otolith microstructure to examination of age and growth of juvenile chum salmon Oncorhynchus keta inhabiting coastal waters, formation of otolith increments was investigated for juveniles reared in a seawater aquarium and in net pens. In all otoliths examined, a distinctive check was formed at the time of sea entry of the fish. The deposition of otolith increments after the check was daily for rearing both in the aquarium (57 days) and in the net pens (26 days). Check formation associated with sea entry was also observed in otoliths of juvenile salmon collected 1 km off the coast of Shari, Hokkaido, Japan. Transmitted light observation of otoliths of those fish revealed a transition in otolith increment appearance from dark to light. Otolith Sr: Ca ratio remarkably changed from a low to a high level, coinciding with the transition in otolith appearance. It is suggested that the transition was associated with individual sea entry. This study demonstrated that the check and/or transition associated with sea entry are applicable to a benchmark for otolith increment counts of juvenile chum salmon inhabiting coastal waters.     

Why do trees decline or dieback after a strong wind? Water status of Hinoki cypress standing after a typhoon

We examined the water status of Hinoki cypress, Chamaecyparis obtusa (Siebold & Zucc.) Endl., trees after a severe typhoon to determine possible causes of the decline and dieback that can occur in what appear, at first, to be healthy trees in typhoon-damaged forest stands. We found that in apparently healthy trees in a storm-damaged stand, the water conducting area of the trunk cross section was greatly reduced compared with that of similarly sized trees in a nearby undamaged stand. Although leaf specific hydraulic resistance (Wl) from soil to leaf and from trunk to leaf was higher in trees from the storm-damaged than the undamaged stand, Wl values from soil to root were similar. Diurnal patterns in the rates of change in trunk diameter differed between trees in the damaged and the undamaged stand. We conclude that increased aboveground hydraulic resistance caused by a reduction in trunk water conducting area could be a major reason for the decline and dieback of apparently healthy trees in typhoon-damaged stands.