Vitrification of fully grown and growing porcine oocytes using germinal vesicle transfer

The survival rate of vitrified germinal vesicle (GV) stage porcine oocytes is very low, and it is not known if the vitrification damages the nucleus, cytoplasm or both. We have evaluated the eventual GV or cytoplasmic damage in fully grown (FG) and growing vitrified oocytes. Fifty-five percent of nonvitrified FG cumulus-denuded oocytes reached the metaphase II (MII) stage in culture. When growing oocytes from preantral (PA) and early antral (EA) follicles were matured in vitro, almost all oocytes were arrested at the GV stage (GV stage: PA 88.9 and EA 79.5%, respectively). When fresh GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes and matured in vitro, some of them reached the MII stage (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). The maturation rate of vitrified FG oocytes was only 6.1% but increased dramatically when vitrified GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes (MII stage: VitFG/FG 43.9%, VitPA/FG 7.1% and VitEA/FG 26.3%, respectively). These results were not significantly different from those for the nonvitrified groups (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). We activated the reconstructed oocytes that received fresh or vitrified GVs (FG/FG, EA/FG, VitFG/FG and VitEA/FG) and examined their embryonic development. Cleaved embryos (nonvitrified groups 13.0-61.8%, vitrified groups 33.3-40.0%) and blastocysts (nonvitrified groups 0.0-18.2%, vitrified groups 0.0-2.9%) were obtained after activation. These results demonstrate that vitrified porcine GVs maintain maturational and developmental competence and that vitrification predominantly damages the cytoplasm.     

Identification of bacteria in total mixed ration silage produced with and without crop silage as an ingredient

As a forage source for total mixed ration (TMR) silage production, locally produced crop silage is now used in addition to imported hay. This type of TMR ensiling is regarded as a two‐step fermentation process; hence, a survey was carried out to determine whether the bacteria in crop silage affect the subsequent TMR ensiling. Fermentation product contents and bacterial community were determined for TMR silage and its ingredient silages collected in August, October and November. August product contained corn, sorghum and Italian ryegrass silages, October product had wheat silage exclusively and November product did not include any crop silages. Acetic acid, lactic acid, 2,3‐butanediol and ethanol were predominant fermentation products in corn, sorghum, Italian ryegrass and wheat silages, respectively. Robust lactic acid fermentation was seen in TMR silage, even if acetate‐type and alcohol‐type silages were mixed as ingredients. The finding that bacterial community of the TMR silage appeared unrelated to those of ingredient silage supported this. Silages of various fermentation types can therefore be formulated without interfering with lactate‐type fermentation in TMR silage.     

Follicular persistence induced by adrenocorticotropic hormone administration in goats

The objective of the present study was to examine the effect of adrenocorticotropic hormone (ACTH) administration on the induction of persistent cystic follicle in the goat in order to establish a method to experimentally induce cystic follicle. Four cross-bred goats were intramuscularly administered ACTH at 0.78 and 6.25 μg/10 kg twice a day from Days 15 to 21 (Day 0 was defined as the day of last estrus). Follicular status in the ovary was monitored by ultrasound examination. The plasma concentrations of estradiol, progesterone and cortisol were measured. Treatment with ACTH at the 0.78 and 6.25 μg/10 kg levels caused persistent follicles (> 10 days delay from the expected ovulation date) in 50% of the goats in both treatment groups. In those animals, ovulation occurred 17 and 27 days and 11 and 12 days after the expected days in the 0.78 and 6.25 μg/10 kg groups, respectively. The maximum follicle diameters were 10 and 9 mm in the 0.78 and 6.25 μg/10 kg ACTH groups, respectively. In the control group, the estradiol concentration increased on Day 18 and remained at a high level for a few days. However, such an increase was not seen in both ACTH groups. The estradiol concentration increased gradually from Days 21 to 27 in the 6.25 μg/10 kg ACTH group. These results suggest the possibility that ACTH induces persistent follicles in goats, which may be related to the delay of the onset of estradiol secretion followed by its maintenance at a high concentration.     

A Preliminary Study of 13C-Phenylalanine and 13C-Dipeptide Breath Tests in Horses

This study aimed to establish a standard dose and sample collection time for ¹³C phenylalanine and ¹³C-Dipeptide breath test in horses. To evaluate dose-dependent effects, healthy horses received 2.5 mg/kg, 5 mg/kg, and 10 mg/kg ¹³C phenylalanine dissolved in 1 ml/kg distilled water and 1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg ¹³C dipeptide dissolved in 2 ml/ kg distilled water. Tmax was observed during the sample collection time. For ¹³C phenylalanine, the standard deviation of Cmax at 5 mg/kg was lower than that of 10 mg/kg. For ¹³C dipeptide, the standard deviation of Tmax was the lowest at 5 mg/kg. This study revealed that an optimal dose for breath tests with ¹³C phenylalanine and ¹³C dipeptide may be 5 mg/kg in horses.     

Seminal plasma lactoferrin but not transferrin reflects gonadal function in dogs

Lactoferrin purified from canine seminal plasma by a three-step chromatography procedure had a molecular mass of 75.2 kDa and cross-reacted with antiserum to equine seminal plasma lactoferrin. Seminal plasma lactoferrin concentrations were determined by a competitive enzyme-linked immunosorbent assay (ELISA) by using rabbit anti-equine lactoferrin antibody and alkaline phosphatase-labeled goat anti-rabbit IgG antibody in 14 normal dogs and found to range from 12 to 197 micro g/ml, with a mean value of 77 +/- 59 micro g/ml (the mean +/- SD). Seminal plasma transferrin concentrations were determined by a sandwich ELISA with goat antibody to canine serum transferrin and alkaline phosphatase-conjugated goat anti-canine transferrin antibody and found to range from 0.32 to 12.6 micro g/m l, with a mean value of 2.44 +/- 3.25 micro g/m l. The lactoferrin concentration significantly correlated with the sperm concentration (r=0.7025, P<0.01), but there was no significant correlation between the seminal plasma transferrin concentration and sperm density. These results indicate that seminal plasma lactoferrin, but not transferrin, reflects gonadal function.     

Malaria in cynomolgus monkeys used in toxicity studies in Japan

Plasmodium spp. protozoa cause malaria and are known to infect humans and a variety of animal species including macaque monkeys. Here we report both our experience with malaria recrudescence in cynomolgus monkeys (Macaca fascicularis) in a toxicity study and the results of a survey on Plasmodium infection in cynomolgus monkeys imported to Japan for laboratory use. A cynomolgus monkey from the toxicity study presented with severe anemia and Plasmodium protozoa in erythrocytes on a thin blood smear and was subsequently diagnosed with symptomatic malaria. In this animal, congestion and accumulation of hemozoin (malaria pigment) in macrophages were noted in the enlarged and darkly discolored spleen. As a follow-up for the experience, spleen sections from 800 cynomolgus monkeys in toxicity studies conducted between 2003 and 2013 were retrospectively examined for hemozoin deposition as a marker of Plasmodium infection. The origin of the animals included Cambodia, China, Indonesia, and Vietnam. Hemozoin deposition was confirmed in 44% of all examined monkeys. Monkeys from Indonesia showed the highest incidence of hemozoin deposition (approx. 80%). A high prevalence of Plasmodium infection in laboratory monkeys was also confirmed with polymerase chain reaction (PCR) by using Plasmodium genus-specific primers. Although Japan is not a country with endemic malaria, it is important to be aware of the prevalence and potential impact of background infection with Plasmodium spp. and recrudescence of symptomatic malaria in imported laboratory monkeys on pharmaceutical toxicity studies.     

Analysis of phylogenetic relationships in Mangifera by restriction site analysis of an amplified region of cpDNA

The phylogenetic relationship of 13 Mangifera species collected in Thailand were examined. Among these species, M. foetida, M. odorata, and M. sylvatica have been cultivated as fruit crops for local markets in Thailand, as well as M. indica (common mango). The other nine wild species also seem to have potential values as rootstocks and breeding materials for tolerance to critical environmental conditions. By restriction fragment length polymorphism (RFLP) analysis of a particular region of cpDNA, these species were classified into two groups. The first group consisted of M. indica and M. sylvatica, and the second group consisted of M. caloneura, M. cochinchinensis, M. collina, M. flava, M. foetida, M. gedebe, M. griffithii, M. macrocarpa, M. oblongifolia, M. odorata, and M. pentandra. The species belonging to the same group are monomorphic for 20 restriction enzymes tested. The present study indicated low diversity in the amplified region of cpDNA among Mangifera species tested, even though the species in the second group were scattered to different subgenera and sections.     

Trophic sources and linkages to support mesozooplankton community in the Kuroshio of the East China Sea

Many migratory fishes reproduce and recruit around the Kuroshio, and their survival of early life stages is supported by mesozooplankton. Mesozooplankton standing stocks and productivity equivalent to those on the continental shelf have been found in the Kuroshio; however, there is limited information on trophic sources and linkages to support the mesozooplankton community. Here, we evaluate mesozooplankton feeding on protists and their trophodynamics importance by removal bottle experiments in the Kuroshio of the East China (ECS‐Kuroshio). Pico‐ and nano‐autotrophs dominated chlorophyll a concentrations throughout the study sites across the continental shelf, within the Kuroshio path and in adjacent waters. Calanoid and poecilostomatoid copepods comprised more than 85% of mesozooplankton biomass. Significant mesozooplankton ingestion rates were found for nano‐autotrophs based on size‐fractionated chlorophyll; for haptophytes, chrysophytes, chlorophytes, and diatoms from pigment‐based phytoplankton analysis; and for naked ciliates in the microzooplankton. Based on the estimates of individual carbon budgets, nano‐autotrophs and naked ciliates ingested by mesozooplankton composed 39% of their food requirements, suggesting other available prey like nano‐heterotrophs. These results imply that mesozooplankton ingestion in the ECS‐Kuroshio has great impacts on protozoan and phytoplankton communities and their major trophic pathways are from nano‐sized auto‐ and heterotrophs and ciliates to copepods.     

Seasonal variations in the concentration of antimicrobial components in milk of dairy cows

The incidence of bovine mastitis and the bulk milk somatic cell count (BMSCC) are influenced by season, which may be associated with innate immune functions, including antimicrobial components in mammary glands. Therefore, the present study was conducted to examine the effect of season on antimicrobial components in milk. Rectal temperature and plasma cortisol, thyroxine, and derivatives of reactive oxygen metabolites (d‐ROMs) were measured as stress parameters. Concentrations of lactoferrin (LF), lingual antimicrobial peptide (LAP), psoriasin (S100A7), and Immunoglobulin A (IgA) in milk were measured as indicators of innate immune function. LF and LAP concentrations were significantly lower in summer than in winter and spring, respectively, whereas the concentration of S100A7 was significantly lower in winter than in spring and autumn. The rectal temperature was significantly higher in summer than in other seasons, whereas plasma cortisol, thyroxine, and d‐ROMs did not exhibit any seasonal variation. In conclusion, even though stress parameters were not changed, the concentration of antimicrobial components, such as LF and LAP, decreased in summer, which may explain the frequent occurrence of mastitis during this season.