Induction of reactive oxygen generation and functional changes in mitochondria and their involvement in the development of bacterial rot in lettuce caused by Pseudomonas cichorii

Pseudomonas cichorii is the major causal agent of bacterial rot disease in lettuce, and apoptosis-like programmed cell death is closely associated with disease development. Depletion of cellular ATP and expression of the alternative oxidase gene was observed in lettuce leaves inoculated with P. cichorii suggesting mitochondrial dysfunction. Cytochemical observation showed production of reactive oxygen species in mitochondria of P. cichorii-inoculated lettuce leaf cells. Release of cytochrome c into the cytosol was also induced by inoculation with the bacterium. Superoxide production was observed in the mitochondrial fraction isolated from P. cichorii-infected leaves much more intense than water-treated leaves. Loss of swelling ability was also observed in the mitochondrial fractions following inoculation with P. cichorii. Intriguingly, inhibitors of the mitochondrial respiratory complex III prevented loss of swelling ability, whereas superoxide generation was scarcely affected by inhibitors of mitochondrial permeability transition in the mitochondrial fractions. Inhibitors of the mitochondrial respiratory complex III and mitochondrial permeability transition delayed not only P. cichorii-induced cell death, but also disease development. In contrast, P. cichorii-induced DNA fragmentation was not inhibited in the presence of either type of inhibitor. The findings suggest that mitochondria may play a crucial role in DNA fragmentation-independent cell death pathway(s).     

Artificial insemination with frozen epididymal sperm in cats

Artificial insemination with frozen cauda epididymal sperm was performed in cats. Sperm were transmigrated from the epididymides in 10 male cats. The mean sperm motility and viability were 67% and 82.5%, respectively, and 11.6 x 10(7) sperm were recovered. The mean sperm motility after thawing was 24.0%. Eleven female cats received unilateral intrauterine insemination of 5 x 10(7) sperm, and the conception rate was 27.3% (3/11). This was the first case of conception obtained with frozen epididymal sperm in cats.     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

Embryo transfer in the cat during the non-breeding season

Studies were conducted to investigate the possibility of embryo transfer in the cat during the non-breeding season. Estrus was induced in 19/22 (86.4%) cats using a porcine pituitary gland preparation. Uterine horns were flushed in 5 cats 6-8 days after mating with expanded blastocysts being collected from 4 cats. One to nineteen blastocysts per cat were transferred to the uterine horns of 6 recipient cats in which ovulation had been induced with HCG. The time differences between time of ovulation in donor and recipient animals were 0.5 days earlier in the recipient (2 cats), 1 day later in the recipient (3 cats), and no difference (1 cat); conception occurred in all the recipients. The ratio of fetuses to transplanted embryos were 1/1, 1/2, 2/3, 2/6, 4/7, and 2/19, respectively. Fetal death occurred in 2 cats at days 22 and 25 and abortion occurred in 3 cats at days 34, 35 and 39. There was a delay in the expulsion of placentae in the animals that experienced fetal death on days 22 and 25, expulsion occurring on days 36 and 56, respectively. One cat was treated with progesterone and carried 2 fetuses to day 66; pregnancy was terminated by cesarean section. In conclusion, it was demonstrated that embryo transfer can be performed in cats in which estrus and ovulation have been induced with porcine pituitary gland preparation during the non-breeding season. However, luteal activity needs to be supplemented by exogenous progesterone administration to maintain pregnancy.     

Effects of prostaglandin F2 alpha-analogue administration on luteal function, implantation of embryos and maintenance of pregnancy in bitches

The present experiment was carried out to clarify the effect of administration of the prostaglandin F2 alpha (PGF2 alpha)-analogue on the luteal function and the maintenance of pregnancy in bitches. Fifty-one bitches received a single inoculation of PGF2 alpha-analogue by intramuscular injection. The effect of this agent was observed by monitoring progesterone (P) levels and the state of the uterus by laparotomy, the occurrence of abortion, and the state of parturition. As a result, when bitches were administered with 100-400 micrograms at the beginning of the luteal phase, the decrease in the P level was temporary. In bitches inoculated with 100-800 micrograms of PGF2 alpha-analogue at the functional luteal stage, the P level began to decrease as early as on the following day after injection. In those treated with 100-200 micrograms of PGF2 alpha-analogue at 10-15 days of pregnancy, pregnancy was maintained in 3 of 5 bitches that had received the treatment at day 10, while in the remaining two, all embryos died after implantation. In those that had received the same treatment at day 15, only 2 of 7 maintained pregnancy. Pregnancy was interrupted in eight bitches treated with doses of 100-200 micrograms at days 25-45. In four bitches treated with doses of 100-200 micrograms at day 55, premature birth was induced after 30-44 hr. In conclusion, regression of the corpus luteum, abortion, and premature birth were induced in bitches treated with 100-200 micrograms at each stage, except the beginning of the luteal phase and of the pregnancy.