Nuclear transfer in cattle using in vivo-derived vs. in vitro-produced donor embryos: Effect of developmental stage

Abstract

To determine the best developmental stage of donor embryos for yielding the highest number of clones per embryo, we compared the efficiencies of nuclear transfer when using blastomeres from morulae or morulae at cavitation, or when using inner‐cell‐mass cells of blastocysts as nuclear donors. This comparison was done both on in vivo‐derived and in vitro‐produced donor embryos. In experiment 1, with in vivo‐derived donor embryos, nuclei from morulae at cavitation supported the development of nuclear transfer embryos to the blastocyst stage (36%) at a rate similar to that of nuclei from morulae (27%), blastomeres from morulae at cavitation being superior (P < 0.05) to inner‐cell‐mass cells from blastocysts (21%). The number of blastocysts per donor embryo was significantly (P < 0.05) higher when using nuclei from morulae at cavitation (15.7 ± 4.1) rather than nuclei from morulae (9.8 ± 5.5) or blastocysts (6.3 ± 3.3). With in vitro‐produced donor embryos (experiment 2), nuclei from morulae yielded slightly more blastocysts (32%) than nuclei from morulae at cavitation (29%), both stages being superior to nuclei from blastocysts (15% development to the blastocyst stage). Morulae at cavitation yielded a higher number of cloned blastocysts per donor embryo (11.5 ± 5.9) than did morulae (9.3 ± 3.2) and blastocysts (3.3 ± 1.4). Transfer of cloned embryos originating from in vivo‐derived morulae, morulae at cavitation, and blastocysts resulted in four pregnancies (10%), three pregnancies (7%), and one (17%) pregnancy on day 45. The corresponding numbers of calves born were 3 (4%), 3 (7%), and 0, respectively. After transfer of blastocysts derived from in vitro nuclear donor morulae (n = 16) and morulae at cavitation (n = 7), two (20%) and two (50%) recipients, respectively, were pregnant on day 45. However, transfer of seven cloned embryos from in vitro donor blastocysts to three recipients did not result in a pregnancy. Using in vitro‐produced donor embryos, calves were only obtained from morula‐stage donors (13%). Our results indicate that the developmental stage of donor embryos affects the efficiency of nuclear transfer, with morulae at cavitation yielding a high number of cloned blastocysts. © 1996 Wiley‐Liss, Inc.

Effect of embryonic cell cycle of nuclear donor embryos on the efficiency of nuclear transfer in Japanese black cattle

“…In the present study, however, we did not observe significant differences in the rates of pregnancy (40 versus 23%), abortion (21 versus 0%) and calving (28 versus 23%) between sixth and seventh cell cycle-stage embryos, due to the small sample sizes. The rate of abortion in the present study (21% for sixth cell cycle stage) was similar to those in previous studies (18 to 34%, Bondioli, 1993; Stice & Keefer, 1993; Zakhartchenko et al ., 1996; Bourhis et al ., 1998).…”

“…Although we did not determine the efficiency of ECNT using the fifth cell cycle (16- to 31-cell)-stage embryos (mean cell number: 24), which can be recovered from the uterus (Newcomb et al ., 1976), we could produce about 20 identical blastocysts if the rate of development of NT embryos to the blastocyst stage was about 80%. Calving rates for the fifth and sixth cell cycle (16- to 63-cell)-stage embryos were reported to be 10 to 26% (Bondioli, 1993; Stice & Keefer, 1993; Zakhartchenko et al ., 1996; Bourhis et al ., 1998). Thus, we could obtain about five calves at most (about 20 blastocysts × 0.26) for the fifth cell cycle (16- to 31-cell)-stage embryos, which was similar to the rate for the sixth cell cycle (32- to 63-cell)-stage embryos (Table 2).…”

“…However, the rate of development to the blastocyst stage of NT embryos originating from the fifth cell cycle (16- to 31-cell)-stage embryos was reported to be lower than that of sixth cell cycle (32- to 63-cell)-stage embryos (Bondioli et al ., 1990). Blastocyst development of NT embryos reconstructed with inner cell mass cells of blastocysts was also lower than that with blastomeres of the sixth cell cycle (32- to 63-cell)-stage embryos (Zakhartchenko et al ., 1996). Hence, the present results indicate that sixth cell cycle (32- to 63-cell)-stage embryos yield the highest number of identical blastocysts (about 20/donor embryo) and calves (about six/donor embryo) in Japanese black cattle.…”

“…It is important to determine the optimum developmental stage of nuclear donor embryos that can be recovered from the uterus of cattle non-surgically for efficient cloning by ECNT. Several studies have examined in vitro and in vivo development of bovine NT embryos derived from 16- and 64-cell-stage embryos (Bondioli et al ., 1990) and morulae and blastocysts (Zakhartchenko et al ., 1996). However, the effect of the embryonic cell cycle of the nuclear donor embryo on the efficiency of ECNT has not been studied in cattle.…”

Effects of the Age of Donor Embryos on the Developmental Ability of Bovine Nuclear Transferred Eggs In Vitro.

“…A small proportion of enucleated oocytes receiving an ICM cell from a sheep [21], bovine [22][23][24], rabbit [25] or mouse (26; Tsunoda et al, unpublished observation) blastocyst has developed to blastocysts in vitro, and all except those receiving rabbit blastocysts have developed to full-term pregnancy. These findings indicate that the nucleus of ICM cells could be reprogrammed at the time of fertilization in enucleated oocytes.…”