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The ultrastructures of the upper surface layer of rat articular cartilage were studied with our "in vivo cryotechnique" followed by freeze-substitution method for scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Rat hip or knee articular cartilage was quickly frozen by the in vivo cryotechnique with liquid isopentane-propane cryogen (-193 degrees C), and surface areas of some frozen specimens were freeze-fractured with a scalpel in liquid nitrogen. They were freeze-substituted and freeze-dried, ion-sputtered, and then observed in SEM. Other frozen specimens were routinely freeze-substituted and embedded in epoxy resin for TEM. Many globular structures were detected in the thick upper surface layer that had not been revealed by the conventional fixation methods. Their sizes were reduced by Triton X-100 treatment, and their localization was also detected in synovial fluid, as revealed by SEM. Such globular lipid-like structures in the upper surface layer of hip or knee articular cartilage might contribute to joint lubrication.

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ABSTRACT. Parentage testing was performed in sixteen litters by canine artificial inseminations with frozen semen from different sires on Days 5 and 7 after the LH surge. It became apparent that only 25% of dams had superfecundation, but 43.8% of dams were whelped after insemination only on Day 5 after the LH surge and 31.3% of dams after insemination only on Day 7. Of the total 87 puppies , 46% were born after insemination on Day 5 after the LH surge and 54% after insemination on Day 7. This result strongly suggested that canine artificial insemination with frozen semen could be sufficiently successful also on Days 5 and 7 after the LH surge. KEY WORDS: canine, optimal time, parentage testing.J. Vet. Med. Sci. 65(9): 1003-1005, 2003 Canine ovulation occurs 48 hr after the LH surge in most bitches [2,13,19]. Ova are fertilizable from 60 hr (2.5 day) to 108 hr (4.5 day) after ovulation [18]. The estimated intrauterine fertile lifespan of frozen-thawed semen is <24 hr [1] and the sperm require 7 hr for capacitation [11]. Therefore, theoretically the optimal time for insemination with frozen semen would be around Day 4 after the LH surge [9] and Days 4 and 7 after the LH surge have previously been recommended [10]. Nishiyama et al. [12] reported that the conception rate from inseminations on Days 5 and 7 after the LH surge was at least twice as high as that from inseminations on Days 4 and 6. This study investigated which day had the higher conception rate when insemination with frozen semen was performed twice, on Days 5 and 7, after the LH surge. To discriminate the individual sire on Days 5 and 7 after the LH surge, frozen semen with a fertile lifespan within 24 hr was used and parentage testing by microsatellite markers was performed.Twenty female and 8 male beagles keeping at the Laboratory of Theriogenology of Nihon University were used for this study. The ages of the bitches and sires were 3.7 ± 1.2 (1.2-5.5) and 3.4 ± 1.4 (1.8-5.8) years, respectively. The animals were maintained in an animal room and housed in individual cages. They were supplied dry food (Science Diet TR , Maintenance, Hill's and Corgate Japan Co., Tokyo, Japan) once a day and drinking water ad libitum.Only the second fraction of ejaculate was collected by manual manipulation for use as frozen semen. The volume, concentration, color, progressive motility and abnormality were estimated in undiluted semen. The semen was centrifuged (500 g, 5 min) and the sperm-rich fraction was extended with modified TRIS-yolk-citrate extender, twice. The number of washed sperm was adjusted to 2 × 10 8 /ml with modified TRIS-yolk-citrate extender at 30°C and cooled to 4°C stepwise at -0.3°C/min in an automatic freezer. The solution was added to the same total volume of 6% glycerol-TRIS-yolk-citrate extender with an additional one-fourth at 10 min intervals, placed in 0.5 ml plastic straws and kept for 90 min for equilibration. The straws were placed horizontally 5 cm above the surface of liquid nitrogen fill in a styrofoam box, for 6 min and subsequently pl...

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