Optimal Timing for Canine Artificial Insemination with Frozen Semen and Parentage Testing by Microsatellite Markers in Superfecundency

Abstract

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...

Individual fertility differences in the frozen‐thawed spermatozoa among semen donors in the Labrador Retriever

“…Of 13 males, 6 males exhibited severely poor fertility (less than 20% pregnancy rate) and 3 males exhibited high fertility (over 70% pregnancy rate) on artificial insemination. Since the optimal time for insemination with frozen semen theoretically appeared to be around day 4 after the LH surge [9], and it has been suggested that artificial insemination with frozen semen is successful on days 4 and 6 [10], and days 5 and 7 [11,12] after the LH surge in canine species, we thus performed transcervical insemination between days 4 and 8 after the estimated LH surge in the Labrador Retriever. However, as shown in Table 2, heterospermic insemination in the present study revealed the optimal timing for transcervical artificial insemination with frozen-thawed semen to be by day 6 after the estimated LH surge.…”

Prediction of the optimal time for insemination using frozen-thawed semen in a multi-sire insemination trial in bitches

“…Furthermore, there is a strong, positive, linear correlation between the interval since the LH peak and the stage of embryonic development, with the first potentially fertilised oocytes and zygote identified 7 d after the LH peak (Bysted et al, 2001). In the light of the findings by Nöthling et al (2003) and Tsumagari et al (2003), this synchrony suggests a need to more closely compare the fertility of Days 5, 6 and 7 after PPC first reaches a value between 6 and 9 nmol/L. Some dog breeders believe that the time of mating or the time of artificial insemination has an effect on the gender ratio of the resulting litters.…”

“…Their bitches yielded mean litter sizes of 4 ± 2.4 (SD), 6.6 ± 2.5 and 6.5 ± 2.5 from the inseminations done 5, 7 and 5 and 7 days after PPC exceeded 6 nmol/L, respectively; suggesting that good fertility is possible from inseminations done 7 days after PPC first exceeded 6 nmol/L. Unfortunately, the study by Tsumagari et al (2003) does not permit a comparison between the fertility of inseminations done on either Day 5 or Day 7 with those done on Day 6. Tsutsui (1975) and Bysted et al (2001) showed that embryonic development is synchronized among embryos within a bitch, suggesting that fertilization occurs over a short period of time within a bitch.…”

“…If such a belief is true, it holds large economic and breeding value to dog breeders and is it worthy of proper characterisation. Insemination with frozen-thawed spermatozoa 5 and 7 days after PPC first exceeded 6 nmol/L yielded no difference in the proportions of male and female pups born (Tsumagari et al, 2003). Ennis and Gallagher (1994) established a method of sexdetermination using the amelogenin locus in cattle, which is also being used in horses and pigs.…”

“…Only two studies (Badinand et al, 1993;Tsumagari et al, 2003) on bitches were, however, of a design suitable to compare the fertility of inseminations with frozen-thawed spermatozoa performed on different days during the same oestrous period of a bitch. Being able to compare the fertility of different days during the same oestrous period is essential to determine the day of the oestrous period on which frozen-thawed spermatozoa will yield the highest fertility which, in turn, is a prerequisite for determining the minimum effective number of inseminations and the minimum effective sperm dose.…”