Successful Production of Piglets Derived from Expanded Blastocysts Vitrified Using a Micro Volume Air Cooling Method without Direct Exposure to Liquid Nitrogen

This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN₂). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN₂ for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN₂.     

Effects of leptin and leptin peptide amide on the release of luteinizing hormone, growth hormone and prolactin from cultured porcine anterior pituitary cells

The present study was carried out to determine whether leptin or leptin (116–130) peptide amide (lep (116–130)), an active fragment of the native protein in rats, is able to stimulate the release of luteinizing hormone (LH), growth hormone (GH) or prolactin (PRL) from cultured porcine anterior pituitary (AP) cells in vitro. The AP cells were obtained from 6 month‐old pigs and were incubated for 3 h with 10^?11?10^?7 mol/L leptin or lep (116–130) after being cultured in Dulbecco's modified Eagle's medium for 3–4 days. Leptin significantly increased the concentration of LH and GH in the culture medium at concentrations of 10^?8 and 10^?7 mol/L, respectively, compared with the controls (P < 0.05). Leptin did not increase the concentration of PRL in the culture medium. In contrast to these results, no effects of lep (116–130) on the release of LH, GH or PRL were seen in the cultured cells. These results suggest that leptin stimulates the release of LH and GH by acting directly on porcine AP cells, and that a fragment of leptin protein comprising amino acids 116–130 is not associated with the secretion of hormones in pigs.     

A numerical analysis of population dynamics of the sand lance (Ammodytes personatus) in the eastern Seto Inland Sea, Japan

The Japanese sand lance (Ammodytes personatus) population in the Seto Inland Sea shows large fluctuations every few years. Its recruitment to the fishery resources is much more dependent on the survival rate of 0-age fish than is the case for other fish species because 0-age fish directly recruit to next-year spawning adults. Since the 0-age fish population depends on its larval population, which is easily influenced by natural and human impacts, high larval mortality due to predation by adults and high dispersion of larvae by wind-driven currents from spawning grounds to other locations can have very important effects on stock fluctuations. A numerical model of population dynamics of the sand lance in the eastern Seto Inland Sea was developed that focuses on wind-induced transport and adult predation of the larvae. The model successfully simulated the two-year periodic fluctuation and was consistent with actual fluctuations in fishery catches. It suggests that the various coefficients given in the model are appropriate to explain the actual population dynamics of the sand lance. The model shows that adult predation on the larvae plays a very important role in the two-year periodic fluctuation, and the maximum predation rate is estimated to be ten individuals per day. Wind-induced transport modifies the impact of this predation. Variances in the two-year periodic fluctuation become smaller with weaker westerly wind velocity. The appropriate transport rates from Bisan Strait and the Harima-nada Sea are estimated to be 0.04 d^-1 and 0.01 d^-1, respectively.     

The synthetic antioxidant, ethoxyquin, adversely affects immunity in tilapia (Oreochromis niloticus)

Ethoxyquin (EQ) has been used as an antioxidant in livestock, aquaculture and pet foods. Animal food safety law has established the upper limit of EQ in animal feed at 150 mg kg⁻¹. However, the risk of EQ at the approved level for aquaculture feed (150 mg kg⁻¹) to fish health is unknown. Here, we examine the effect of EQ on the immunity of tilapia ( Oreochromis niloticus ). EQ concentration in the blood reached 0.16 mg L⁻¹ in fish fed EQ at the approved level. This level of EQ inhibited phagocytic activity of leucocytes in vitro and antibacterial activity of whole blood in vivo . Furthermore, pyknosis in the liver was observed throughout the duration of feeding. However, after 30 days of experimental challenge with feed containing 150 mg kg⁻¹ of EQ, no significant difference was observed in mortality. Although EQ at the approved level in feed causes immunosuppression in fish, the severity of immunosuppression does not lead to a lowering of disease resistance for short feeding periods.     

Effect of sevoflurane concentration on visual evoked potentials with pattern stimulation in dogs

The purpose of this study was to investigate the effects of sevoflurane concentration on canine visual evoked potentials with pattern stimulation (P-VEPs). Six clinically normal laboratory-beagle dogs were used. The minimum alveolar concentration (MAC) of sevoflurane was detected from all subjects by tail clamp method. The refractive power of the right eyes of all subjects was corrected to −2 diopters after skiascopy. For P-VEP recording, the recording and reference electrode were positioned at inion and nasion, respectively, and the earth electrode was positioned on the inner surface. To grasp the state of CNS suppression objectively, the bispectral index (BIS) value was used. The stimulus pattern size and distance for VEP recording were constant, 50.3 arc-min and 50 cm, respectively. P-VEPs and BIS values were recorded under sevoflurane in oxygen inhalational anesthesia at 0.5, 1.0, 1.5, 2.0, 2.5 and 2.75 sevoflurane MAC. For analysis of P-VEP, the P100 implicit time and N75-P100 amplitude were estimated. P-VEPs were detected at 0.5 to 1.5 MAC in all dogs, and disappeared at 2.0 MAC in four dogs and at 2.5 and 2.75 MAC in one dog each. The BIS value decreased with increasing sevoflurane MAC, and burst suppression began to appear from 1.5 MAC. There was no significant change in P100 implicit time and N75-P100 amplitude with any concentration of sevoflurane. At concentrations around 1.5 MAC, which are used routinely to immobilize dogs, sevoflurane showed no effect on P-VEP.     

Chronic spinal epidural abscess in a cat: a case report with an unusual imaging finding

A 1-year-old domestic shorthair cat was evaluated for a chronic history of back pain, dysuria, and paraplegia. Radiographic and computed tomographic examinations showed circumferential widening of the vertebral canal at T13 and T14. A spinal epidural abscess (SEA) compressing the spinal cord from the level of T11 to L1 was suspected following intravenous contrast administration, and was confirmed by surgical exploration and histopathological analysis. The cat recovered its motor and bladder functions following surgical decompression and antibiotic therapy. SEA is a neurological emergency requiring prompt treatment. However, the present case had a prolonged disease course and pressure atrophy of the vertebrae was strongly suspected. To our knowledge, this imaging finding has not been reported in dogs or cats with SEA.     

Effects of adding luteinizing hormone to a medium containing follicle stimulating hormone on progesterone-induced differentiation of cumulus cells during meiotic resumption of porcine oocytes

In the present study, we investigated the effects of adding luteinizing hormone (LH) to a medium containing follicle stimulating hormone (FSH) on the shift in expression of progesterone receptor (PR) isoforms (PR‐A and PR‐B) and the roles in function of cumulus cells of cumulus‐oocyte complexes (COC). The level of PR‐B mRNA in cumulus cells was up‐regulated by FSH during the first 16‐h cultivation but the level was significantly decreased at 20 h. The decrease of PR‐B mRNA was accelerated when COC were cultured with FSH and LH. Still, a high level of total PR mRNA was maintained in cumulus cells cultured with or without the addition of LH up to 20 h, suggesting that the expression of PR isoforms was shifted from PR‐B to PR‐A in cumulus cells. The reduction of PR‐B was also induced by addition of progesterone to FSH‐containing medium. The addition of LH or progesterone to FSH‐containing medium stimulated cumulus expansion of COC as compared with that of COC cultured with FSH. In the expanded COC, ADAMTS‐1 which is expressed in granulosa cells and cumulus cells in rodent follicles through LH‐induced progesterone‐ and PR‐dependent pathway, was more accumulated within the COC matrix. These results suggest that the addition of LH or progesterone to FSH‐containing medium is required for the differentiation of cumulus cells, such as cumulus expansion, mediated by the shift from PR‐B to PR‐A in them.     

Demographic Analysis of Breeding Structure in Japanese Thoroughbred Population

To investigate the breeding structure in the Japanese Thoroughbred population, we applied a demographic analysis to the populations of foals produced from 1978 to 2005. The migration rate estimated from the proportion of foals produced by imported breeding horses was around 40% over the investigated period. After early 1990s, the migration rate through stallions imported from USA sharply increased. The average generation interval was within range of 10.5–11.5 years. The longer generation interval of Thoroughbred was considered to be a reflection of the fact that Thoroughbred horses begin breeding only after completing their performance in races. After the peak of 729 in 1993, the number of sires of foals progressively declined to 358 in 2005. Although the coefficient of variation of the progeny number of sires was within range of 1.0–1.2 until early 1990s, it gradually increased and reached the value of 1.6–1.7 in recent years. The effective number of sires consistently decreased after the peak of 302.6 in 1992, and reached 120–130 in recent years, which is 25–30% of the actual number of sires. In parallel, the demographic estimate of the effective population size declined after early 1990s. The main cause of the observed change in the breeding structure was inferred to be the intensive use of a limited number of stallions for breeding.     

Influence of after-ripening environments on the germination characteristics and seed fate of Italian ryegrass (Lolium multiflorum)

Italian ryegrass ( Lolium multiflorum Lam.) is an exotic winter annual weed that recently has seriously infested wheat fields in central Japan. To understand the influence of tillage systems on the germination ecology and seedbank dynamics of naturalized Italian ryegrass, we compared the fate of the seeds that were after-ripened in the soil with that of the seeds on the soil surface, and then examined the germination characteristics of the seeds retrieved from both treatments by germination tests in both a light/dark (12 h photoperiod) condition and in constant darkness. The seedling emergence was greater initially for the seeds after-ripened on the soil surface than for the seeds in the soil. The component of emergent seeds after-ripened on the soil surface increased as time passed during the summer, whereas that of emergent seeds in the soil scarcely increased. This contrasting emergence was, in part, attributable to the ability of the seeds to germinate at higher temperatures in the light/dark condition than in constant darkness. These results suggest that untilled systems, which leave many seeds on the soil surface, might promote the rapid depletion of Italian ryegrass seeds through germination before wheat sowing in early winter. In contrast, seed burial by tillage might inhibit the germination of the seeds and form a large transient seed bank available to germinate during the wheat-growing season.