Establishment of a novel equine cell line for isolation and propagation of equine herpesviruses

In the present study, an equine-derived cell line was established by transfecting primary fetal horse kidney (FHK) cells with expression plasmid encoding simian virus 40 (SV40) large T antigen and then cloning them by limiting dilution. The cloned cell line, named FHK-Tcl3, grew well and could be propagated over 30 times by splitting them 1:3. Equine herpesvirus (EHV)-1 and EHV-4 replicated well in FHK-Tcl3. EHV-2 and EHV-4 were isolated from samples collected from horses in the field using FHK-Tcl3, and EHV-3 also propagated in FHK-Tcl3. These results indicated that this novel cell line, FHK-Tcl3, can be used for isolation and propagation of equine herpesviruses.     

Developmental competence of cat oocytes from ovaries stored at various temperature for 24 h

We conducted two experiments to investigate the effects of storage temperature and period for cat ovaries on the meiotic and developmental competence of oocytes collected from the ovaries. In Experiment 1, ovaries were stored in physiological saline for 24 h at 4 C, 23-25 C, or 38 C (cold, room, and incubator temperature groups, respectively), and then oocytes were collected from the ovaries in each group. Morphologically intact oocytes were then selected and cultured in maturation medium for 24 h. Significantly more oocytes reached metaphase II (MII) in the cold temperature group (53.4%) than in the room and incubator temperature groups (20.0 and 2.4%, respectively). In Experiment 2, ovaries were stored in physiological saline at room temperature for 0, 6, 12 or 18 h, and then they were stored at 4 C (cold storage) until reaching a total storage period of 24 h. After storage of the ovaries, morphologically intact oocytes were matured, fertilized with frozen-thawed spermatozoa, and cultured in vitro. The rates of morphologically intact oocytes obtained from the ovaries stored at room temperature for 0, 6, 12 or 18 h were 35.3, 30.0, 26.4 and 14.7%, respectively, and the rates of intact oocytes that reached MII were 63.2, 36.4, 26.5 and 11.9%, respectively. The results suggested that the numbers of morphologically intact oocytes and intact oocytes that reached MII after in vitro maturation decrease gradually as the period of storage at room temperature before cold storage increases. Only oocytes from ovaries stored for 6 h developed to the blastocyst stage after in vitro maturation and fertilization when ovaries were stored at room temperature before cold storage. These results indicate that 24 h storage of ovaries at high temperatures (>23 C) decreases the meiotic competence of oocytes. The quality and developmental competence of oocytes are influenced by the length of storage at room temperature before cold storage.     

A Japanese Black breeding herd exhibiting low blood urea nitrogen: A metabolic profile study examining the effect on reproductive performance

Ten reared cows of a Japanese Black cattle herd in Kagoshima prefecture, Japan, exhibited extremely low blood urea nitrogen (BUN) concentration (2.6 ± 0.6 mg/dL). Examination of dietary feed nutrition and relevant pastureland soil content suggested a correlation with crude protein (CP) deficiency or unbalanced nutritional dietary feeds. Thirteen months after the introduction of a dietary remedial measure (bean cake supplementation), BUN, total cholesterol and albumin concentration from five of the original 10 cows increased significantly compared with their values of before the dietary remedy. The postpartum day open period was significantly lower after the dietary remedial measure than that before it. The abnormally low BUN levels of the cattle herd may be due to inadequate dietary nutritional content, primarily from the imbalance of total digestible nutrient and CP of the feed and far lower han average CP value. In conclusion, routine examination of serum biochemical parameters in Japanese Black breeding cattle may be a useful strategy for determining subclinical metabolic failure of cattle herds, and consequently, its effect on reproductive performance of the herd.     

Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks

This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long‐term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5‐SM and 15‐SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5‐SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5‐SM and 15‐SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5‐SM group showed similar rates of fertilization and blastocyst formation in the fresh non‐stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5‐SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.     

Retort beef aroma that gives preferable properties to canned beef products and its aroma components

The objective of this study is to identify the properties and responsible compounds for the aromatic roast odor (retort beef aroma) that commonly occurs in canned beef products and could contribute to their palatability. The optimal temperature for generating retort beef aroma was 121°C. An untrained panel evaluated both uncured corned beef and canned yamato‐ni beef and found that they had an aroma that was significantly (P < 0.01) similar to the odor of 121°C‐heated beef than 100°C‐heated beef. The panel also noted that the aroma of 121°C‐heated beef tended to be (P < 0.1) preferable than that of 100°C‐heated beef. These results suggest that retort beef aroma is one constituent of palatability in canned beef. GC‐MS (gas chromatography‐mass spectrometry) analysis of the volatile fraction obtained from 100°C‐ and 121°C‐heated beef showed that the amounts of pyrazine, 2‐methylpyrazine and diacetyl were higher in the 121°C‐heated beef than in the 100°C‐heated beef. GC‐sniffing revealed that the odor quality of pyrazines was similar to that of retort beef aroma. Therefore, pyrazines were suggested to be a candidate responsible for the retort beef aroma. Analysis of commercial uncured corned beef and cured corned beef confirmed the presence of pyrazine, 2‐methylpyrazine and 2,6‐dimethylpyrazine.     

Effect of replacement of pyruvate/lactate in culture medium with glucose on preimplantation development of porcine embryos in vitro

The effect of glucose supplementation at different times in in vitro culture on the developmental competence of in vitro produced (IVP) porcine embryos was examined. In Experiment 1, when IVP embryos were cultured in modified NCSU-37 supplemented with pyruvate and lactate (IVC-pyr/lac) for 0 h, 24 h, 48 h, 72 h, 96 h, or 118 h and subsequently in modified NCSU-37 supplemented with glucose (IVC-glu) until Day 6 (Day 0=day of in vitro fertilization), the rates of blastocyst formation were significantly higher in embryos cultured in IVC-pyr/lac for 24 or 48 h (24.4% and 23.0%, respectively) than in embryos cultured in IVC-pyr/lac for the whole culture period (14.5%). However, there were no significant differences between embryos obtained after the energy source replacement and embryos cultured in IVC-glu for the whole culture period on the rates (15.2%-24.4%, and 16.8% respectively). Replacement of pyruvate/lactate with glucose at 58 h of culture in Experiment 2 significantly enhanced the rate (31.3%) compared to those after replacement at 48 h, 53 h and 63 h of culture (20.6%, 20.8%, and 21.1%, respectively). In conclusion, replacement of pyruvate/lactate with glucose as the energy substrate was optimal at 58 h of culture for the development of porcine embryos to the blastocyst stage.     

Effects of ovary storage time and temperature on DNA fragmentation and development of porcine oocytes

The present study was conducted to investigate the effects of storage time and temperature of porcine ovaries on the quality and nuclear maturation in vitro of oocytes obtained from stored ovaries and their subsequent development after in vitro fertilization. The ovaries were stored in physiological saline for 0, 3, 6, 9 and 12 h at various temperatures (4, 15, 25 and 35 C). The pH of follicular fluid obtained from the ovaries, DNA fragmentation of the oocyte nucleus and meiotic competence of oocytes were examined. Some oocytes from ovaries stored at 15, 25 and 35 C for 6 h were fertilized in vitro, and then cultured for 7 days to examine the ability of embryos to develop to the blastocyst stage. When the ovaries were stored at 35 C, the pH of follicular fluid decreased and the proportions of oocytes with DNA fragmented nuclei increased as the storage time was prolonged, and the storage of ovaries for 6, 9 and 12 h resulted in lower maturation rates of oocytes. When the ovaries were stored at 4, 15, 25 and 35 C for 6 h, the storage at higher temperatures (> or =15 C) decreased the pH of follicular fluid and induced nucleic DNA fragmentation in higher proportions of oocytes. None of the oocytes from ovaries stored at 4 C reached metaphase II. The storage of ovaries at 15 C reduced the rates of in vitro fertilized oocytes and subsequent embryo development, but there were no significant differences in the rates of fertilization and blastocyst formation between oocytes from ovaries stored at 25 C and 35 C. Our findings indicate that the storage of ovaries at 25-35 C for 6 h is effective for maintaining the developmental competence of porcine oocytes even though the development rates were lower than those of ovaries stored at 35 C for 3 h.     

Effect of maturation culture period of oocytes on the sex ratio of in vitro fertilized bovine embryos

It has been suggested that the maturational stage of oocytes at time of insemination influences the sex ratio of resulting embryos. However, there are very few reports concerning the relationship between the maturation culture period of oocytes and the sex ratio of resulting embryos. The objective of this study was to investigate the effects of in vitro maturation culture period for bovine oocytes on the sex ratio of in vitro produced blastocysts using a novel technique of loop-mediated isothermal amplification (LAMP). Cumulus-oocyte complexes were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (16, 22, 28, and 34 h). After maturation culture for each period, the oocytes were inseminated with frozen-thawed spermatozoa, and then cultured in vitro. Blastocysts were harvested on Day 7 after insemination, and the sex of the embryos was examined using the LAMP method. The rates of oocytes matured to the metaphase II stage were significantly lower (P < 0.05) in the 16-h maturation group than in the other groups. The proportion of blastocyst formation after insemination was significantly higher (P < 0.05) in the 22-h maturation group than in the other groups. The proportion of male blastocysts increased with the increase in maturation culture period. The proportion of male blastocysts derived from oocytes matured for 34 h was significantly higher (P < 0.05) than from oocytes matured for 16 and 22 h. These results indicate that the sex ratio of in vitro fertilized embryos is apparently influenced by the maturation culture period of the oocytes.     

Retrospective surveillance of metabolic parameters affecting reproductive performance of Japanese Black breeding cows

This retrospective study was conducted to confirm the relationship between pre- and postpartum metabolic parameters and postpartum reproductive performance and to clarify seasonal characteristics of the metabolic parameters by using our metabolic profile test (MPT) database of Japanese Black breeding herds. In evaluation 1, MPT databases of blood samples from multiparous cows collected prepartum and postpartum were divided into two groups according to calving interval, and each MPT parameter was compared. In evaluation 2, the same MPT databases used in evaluation 1 were divided into two groups according to the sampling period. Significant differences were found in the prepartal total protein and postpartal γ-glutamyltransferase in evaluation 1. In evaluation 2, significant differences were found in the prepartal and postpartal total protein, albumin/globulin ratio, and glucose. Clear seasonal differences in MPT results emphasized the usefulness of the MPT in breeding cattle herds fed home-pasture roughage and suggest that unsatisfactory reproductive performance during hot periods reflects inadequate nutritional content of the diet and possible reduced feed intake due to heat stress.     

Effects of long‐term in vitro exposure of ejaculated boar sperm to zearalenone and α‐zearalenol in sperm liquid storage medium

The effects of in vitro exposure of porcine spermatozoa to zearalenone (ZEN) and α‐zearalenol (α‐ZOL) were studied by evaluating several parameters of an in vitro fertilization (IVF) system. For this purpose, boar spermatozoa cultured with semen storage medium containing 0 (control), 10 and 1000 µg/L of ZEN and α‐ZOL for 1 week at 5°C were used for IVF of in vitro matured oocytes. Overall, there were no significant differences in the rates of total penetration, monospermic fertilization, and polyspermic fertilization of oocytes inseminated with spermatozoa from the different groups. Similarly, ZEN and α‐ZOL at 10 and 1000 µg/L did not have detrimental effects on the cleavage and development to blastocysts of oocytes after in vitro fertilization. Although the motility, viability, and plasma membrane integrity of spermatozoa significantly decreased after 3 weeks of storage compared to non‐stored spermatozoa (P < 0.05), ZEN and α‐ZOL at the evaluated concentrations did not exert detrimental effects on the above parameters, even after 3 weeks of storage. These results indicate that prolonged exposure of boar spermatozoa to ZEN and α‐ZOL up to 1000 µg/L under reduced metabolic conditions does not affect their in vitro function.