http://directory.umm.ac.id/Data%20Elmu/jurnal/A/Animal%20Reproduction%20Science/Vol60-61.Issue1-4.Jul2000/1978.pdf

Current state in biotechnology in canine and feline reproduction

Successful artificial insemination in the dog requires good timing of the insemination, skilled collection and handling of the semen, and mastering of insemination techniques. The bitch should be inseminated late in estrus. The insemination dose should contain at least 150 to 200 x 10(6) spermatozoa. Fresh semen can be inseminated vaginally, whereas frozen-thawed semen should be inseminated into the uterus. Pregnancy rates of 84% with fresh semen and 69% with frozen semen are reported.

Achieving canine pregnancy by using frozen or chilled extended semen.

Assisted reproductive technology in canid species.

Early development in canine species follows a very specific pattern. Oocytes are ovulated at the germinal vesicle stage and meiotic resumption occurs in the oviduct. However, because of difficulties in the accurate determination of ovulation time and in the observation of oocyte nuclear stage by light microscopy, these early events have not been fully described. Moreover, the oocyte stage at which sperm penetration occurs is still uncertain since fertilization of immature oocytes has been reported in vivo and in vitro. The aim of this study was to establish the exact timing of in vivo meiotic resumption, fertilization and early embryo development in the bitch with reference to ovulation. Ovulation was first determined by ultrasonography, artificial inseminations were performed daily and oocytes/embryos were collected between 17 and 138 h after ovulation. After fixation and DNA/tubulin staining, the nuclear stage was observed by confocal microscopy. Of the 195 oocytes/embryos collected from 50 bitches, the germinal vesicle stage was the only one present until 44 h post-ovulation, and the first metaphase II stage was observed for the first time at 54 h. Sperm penetration of immature oocytes appeared to be exceptional (three out of 112 immature oocytes). In most cases, fertilization occurred from 90 h post-ovulation in metaphase II oocytes. Embryonic development was observed up to the eight-cell stage. No significant influence of bitch breed and age on ovulation rate, maturation and developmental kinetics was observed. However, some heterogeneity in the maturation/development process was observed within the cohort of oocytes/embryos collected from one bitch. In conclusion, the most peculiar aspect of the canine species remains oocyte meiotic maturation whereas fertilization follows the same pattern as in other mammals.

/pdf/in-vivo-meiotic-resumption-fertilization-and-early-embryonic-28nfenrj2l.pdf

In vivo meiotic resumption, fertilization and early embryonic development in the bitch.

Somatic cell nuclei of giant pandas can dedifferentiate in enucleated rabbit ooplasm, and the reconstructed eggs can develop to blastocysts. In order to observe whether these interspecies cloned embryos can implant in the uterus of an animal other than the panda, we transferred approximately 2300 panda-rabbit cloned embryos into 100 synchronized rabbit recipients, and none became pregnant. In another approach, we cotransferred both panda-rabbit and cat-rabbit interspecies cloned embryos into the oviducts of 21 cat recipients. Fourteen recipients exhibited estrus within 35 days; five recipients exhibited estrus 43‐48 days after embryo transfer; and the other two recipients died of pneumonia, one of which was found to be pregnant with six early fetuses when an autopsy was performed. Microsatellite DNA analysis of these early fetuses confirmed that two were from giant panda-rabbit cloned embryos. The results demonstrated that panda-rabbit cloned embryos can implant in the uterus of a third species, the domestic cat. By using mitochondrial-specific probes of panda and rabbit, we found that mitochondria from both panda somatic cells and rabbit ooplasm coexisted in early blastocysts, but mitochondria from rabbit ooplasm decreased, and those from panda donor cells dominated in early fetuses after implantation. Our results reveal that mitochondria from donor cells may substitute those from recipient oocytes in postimplanted, interspecies cloned embryos. developmental biology, early development, embryo, implantation, pregnancy

https://bioone.org/journals/Biology-of-Reproduction/volume-67/issue-2/biolreprod67.2.637/Interspecies-Implantation-and-Mitochondria-Fate-of-Panda-Rabbit-Cloned-Embryos1/10.1095/biolreprod67.2.637.pdf

Interspecies implantation and mitochondria fate of panda-rabbit cloned embryos.

The intravaginal artificial insemination was carried out in bitches with fresh semen and the number of sperms and the quantity of semen required for conception was examined. Bitches were inseminated with 1 ml (group 1) or 3 ml (group 2) of semen. Each group was divided into 4 subgroups (A-D) inseminated with semen containing 2.0, 1.0, 0.5 or 0.25×108 viable sperms, respectively. The conception rates in groups A, B, C and D were 7/8 (89%), 5/15 (33%), 6/13 (46%) and 0/8 (0%), respectively, and in naturally mated group, 18/19 (95%). Groups B, C and D were significantly different in the rate of conception from the control and group A. No significant difference was observed among groups in the number of puppies born. In case of intravaginal artificial insemination with fresh semen in bitches, semen should contain 2.0×108 sperms/ml to obtain a similar rate of conception and number of offspring as in naturally breeding bitches.

Artificial insemination with fresh semen in beagle bitches

Studies were made on the number of sperms required for fertilization of ova ovulated from the bilateral ovaries in bitches. The experiment was carried out with 44 female beagles, which were divided into four groups of seven to eleven bitches each. The four groups were inseminated with 40 X 10(6) sperms/0.2 ml, 20 X 10(6) sperms/0.1 ml, 10 X 10(6) sperms/0.1 ml, and 3-5 X 10(6) sperms/0.1 ml, respectively. All the bitches were laparotomized at an optimum time for mating and inseminated at the tip of the horn of an unilateral uterus. In the four groups conception took place at a rate of 7/7, 8/8, 10/11, and 2/7, respectively. The number of puppies exceeded the number of ova ovulated on the inseminated side at a rate of 5/7, 6/8, 0/10, and 0/2 in the four groups, respectively. Therefore, it was clarified that when more than 20 X 10(6) sperms/0.1 ml were inseminated in the ipsilateral uterine horn, ova ovulated on the contralateral side were fertilized also. Then a unilateral ovary was ovariectomized in 11 bitches, which were inseminated with 10 X 10(6) sperms/0.1 ml in the ipsilateral uterine horn. As a result, only one bitch became conceptive and gave birth to only one puppy. From these results, it was considered that 10 X 10(6) sperms/0.1 ml may be most adequate for the ova on the side of insemination alone to be fertilized.

Relationship between the number of sperms and the rate of implantation in bitches inseminated into unilateral uterine horn

An experimental trial on embryo transfer in the dog.

Dogs, cats, and pigs have a bicornuate uterus, and transuterine migration of embryos occurs in 40% or more of pregnant animals. However, the mechanism of the transuterine migration has not been elucidated in dogs. Thus, we investigated the occurrence of transuterine migration of embryos when embryos were retained in an unilateral uterine tube with more ovulated ova (Experiment 1), when one ovary was excised (Experiment 2), and when ova ovulated from the right and left ovaries were fertilized with sperm from male dogs with different blood types (Experiment 3). Transuterine migration of embryos was observed in 7/8 (87.5%), 10/10 (100%), and 11/17 (67.4%) fertilized animals in Experiments 1, 2, and 3, respectively. In Experiment 3, intrauterine embryo mixing reported in pigs did not occur. These findings suggest that transuterine migration of embryos occurs due to the number of embryos that enter the uterus but that differences in the number of ovulated ova between the right and left ovaries or the number of embryos retained in the uterine tube do not affect the migration.

Toshimitsu Shimizu

Papers

Artificial insemination with fresh semen in beagle bitches

Relationship between the number of sperms and the rate of implantation in bitches inseminated into unilateral uterine horn

An experimental trial on embryo transfer in the dog.

Factors affecting transuterine migration of canine embryos.

Incidence for transuterine migration of embryos in the dog.