Production of piglets from in vitro-produced embryos following non-surgical transfer

The objective of this study was to enhance procedures for producing piglets derived from in vitro-produced (IVP) pig embryos by non-surgical embryo transfer (ET). The effects of insertion length for the catheter, asynchrony between the age of donor IVP blastocysts and the recipient estrous cycle, and volume of transfer medium were investigated. The IVP blastocysts at 5 days after in vitro fertilization were placed into porcine zygote medium (PZM)-5 supplemented with 10% (v/v) fetal bovine serum (PZM+FBS) in a 0.25 mL plastic straw (21-40 blastocysts per straw) and then transferred into one uterine horn of recipients using the Takumi(?) catheter for deep intrauterine insertion. Successful production of piglets derived from IVP embryos was achieved following non-surgical ET when the catheter was inserted at more than 30 cm anterior to the spiral guide spirette. The efficiency of piglet production (percentage number of piglet(s) born based on the number of embryos transferred) was greater (P<0.05) in recipients whose estrous cycle was asynchronous to that of donors with a 1-day delay (8.3%) than in those with a 2-day (1.5%) or 3-day (0.9%) delay, while pregnancy and farrowing rates (10-40%) did not differ among treatments. When blastocysts were transferred into recipients with 1.0 or 2.5 mL PZM+FBS, there were no significant differences in farrowing rate (30-40%) or average litter size (4.5-6.7) between treatments. The results of the present study indicate that the insertion length of the deep intrauterine catheter and the degree of asynchrony between donor embryos and recipient estrous cycle influenced on pregnancy and birth outcome following non-surgical transfer of IVP blastocysts.     

The effect of partial weaning on the rebreeding performance of primiparous Hereford heifers

One or two 6–7 or 9–10 day old blastocysts were transferred to 39 pure-bred or cross-bred Charolais heifer recipients.The embryos were collected after slaughter of the donors. An insemination catheter (Cassou gun) was used to transfer the blastocysts through the cervix into the uterus. Placement was approximately 5cm beyond the body of the uterus.Pregnancy was determined by slaughter of the recipients on the 45th day of gestation. Nineteen of the 39 recipients were pregnant. The survival rate of embryos transferred was as follows: 6–7 day old embryos: 7 out of 23 (30.4%); 9–10 day old embryos: 18 out of 36 (50%). All embryos: 25 out of 59 (42.3%). The difference between the 6–7 day old embryos and the 9–10 day old embryos was not significant. Twelve of the 20 recipients receiving a bilateral transfer were pregnant, and 6 were carrying twins. This is a satisfactory technique for use in field conditions for the transfer of bovine blastocysts.     

Relationship between plasma progesterone concentrations and pregnancy rates in cattle receiving either fresh or previously frozen embryos

Blood samples were collected for the measurement of progesterone concentrations from 320 Holstein-Friesian heifers on Days 7 and 21 post-estrus. All animals were the recipients of either a fresh or previously frozen embryo on Day 7 and were palpated for pregnancy on Day 60 post-estrus. At the time of transer, progesterone levels were highly variable and were not strongly related to subsequent pregnancy status. There was a tendency for lower pregnancy rates in heifers receiving fresh embryos if progesterone levels were less than 1 ng/ml (33 vs 64% overall), and for previously frozen embryos when progesterone concentrations were less than 3 ng/ml (34 vs 44% overall). Progesterone concentrations were not related to subjective evaluation of corpus luteum quality by palpation per rectum. No heifers which maintained pregnancy had progesterone levels less than 1 ng/ml on Day 21. Only 41% of 247 heifers receiving either fresh or previously frozen embryos that were not pregnant on Day 60 had progesterone concentrations less than 1 ng/ml on Day 21. These data suggest that many recipients that do not maintain a pregnancy will experience an extended estrous cycle after transfer.     

Transuterine embryo migration in recipient cattle

Transuterine migration of bovine embryos following fertilization in vivo is apparently rare, but little is known about migration following embryo transfer. We studied heifers receiving either 1 or 2 in vitro produced embryos to determine 1) the incidence of transuterine migration, 2) the timing of migration and 3) the random or systematic occurrence of the event. In 4 experiments, 436 heifers received embryos and 218 of these were pregnant at necroscopy on either Day 14, Day 18, Day 26 or Day 60 of pregnancy. Overall, 43/218 (20%) of the heifers had embryos that had migrated. The frequency of migration was higher in twin (30/68) than in single (13/150) embryo transfers of pregnant recipients (44 vs 9%; P<0.001), and in contralateral (9/15) than in ipsilateral (33/170) transfers (60 vs 19%; P<0.001). Among the heifers that received embryos by ipsilateral transfer, the migration rate was similar to that in heifers pregnant with a singleton after the transfer of either 1 (2/48) or 2 (4/60) embryos (4 vs 7%, NS). The migration rate was highest at Day 26 (12/37) in heifers receiving twin embryos by ipsilateral transfer but was similar at all other stages of pregnancy (15/111, 32 vs 14%; P<0.01). Migration was first observed by Day 14, and it appears that either further migration occurred over the next 12 d or that migration was associated with a higher survival rate from Day 14 to Day 26. The low migration rate evident at Day 60 suggests that migration by Day 26 was associated with increased embryo or fetal death by Day 60. The data suggest that embryo migration is probably independent for each of a pair of surviving embryos. We conclude that in cattle embryo migration is embryo-dependent, but this capability is dormant unless more than 1 embryo is present in a uterine horn or the embryos are transferred to the contralateral uterine horn. The relationship between migration and embryo survival remains unclear.     

Embryo Transplantation in Cattle

Previous attempts to transfer embryos non-surgically to heifers have been rather discouraging. The present experiment describes a simple non-surgical technique where the 6½–7½ -day old embryo is placed in a 0.25 ml ministraw, fitted into a common insemination gun and transferred cervically to synchronized lactating dairy cows. Thirty-two viable embryos were transferred to the middle part of the uterine horn ipsilateral to the ovary containing the corpus luteum. It was easy to pass the cervix, and most embryos could be deposited in the horn within 1 or 2 min. Eighteen animals were diagnosed pregnant (56% ). It is conceivable that the easy atraumatic transfers and good management (feeding, oestrus control) of the recipients contributed to the high pregnancy rate.     

Repeat superovulation, non-surgical embryo recovery, and surgical embryo transfer in transgenic dairy goats

We investigated the capability of repeat superovulation and non-surgical embryo retrieval, coupled with surgical embryo transfer, to expedite the production of transgenic progeny from transgenic founder dairy goat does. In addition, we compared embryo yields, number of embryos transferred per recipient, pregnancy rates, and offspring born during both the traditional (September-December) and non-traditional (January-May) breeding seasons. Although there were no significant differences, there were numerically more transferable embryos recovered per flush (3.5+/-0.9 vs. 2.4+/-0.9 embryos; mean+/-S.E.M.) and increases in both the proportion of recipients that were pregnant (83 vs. 69% pregnant) and offspring born from total embryos transferred (67 vs. 53% offspring) during the traditional versus the non-traditional breeding season. The transfer of one, two or three embryos did not significantly affect the proportion of pregnant recipients during either season. However, there was a difference (P<0.05) in the proportion of offspring produced for one versus two embryo transfers (89 vs. 44% offspring, respectively) during the non-traditional breeding season. Overall, 14 transgenic offspring were produced from 54 total offspring born, and the kidding interval was reduced to <3 months for six of the seven transgenic does. In summary, repeat superovulation and non-surgical embryo retrieval, coupled with surgical embryo transfer, expedited the production of progeny from transgenic founder does.     

Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis): a retrospective study

The objectives of this study were to determine the pregnancy rate and factors affecting it following nonsurgical embryo transfer in buffalo. Donor buffalo were superovulated with FSH, and embryos collected nonsurgically were evaluated for stage of development and quality. They were transferred nonsurgically to 91 recipients on Days 5 to 7 of the natural (n = 52) or induced (n = 39) estrus (estrus = Day 0). The overall pregnancy rate of 24/91(26.4%) was higher than in earlier reports for buffalo but was much lower than in cattle. Pregnancy rates were not affected by season (autumn vs winter), side of transfer (right vs left uterine horn), or type of estrus (spontaneous vs induced). The pregnancy rate was high 11/27(40.7%) when donors and recipients were closely synchronized, while it was compromised when recipients were in estrus at +12 h (1/7, 14.3%) and at -12 h (5/27, 18.5%). Asynchrony beyond 12 h on either side resulted into conception failure. The pregnancy rate tended to increase with the increase in CL size of recipients, while stage of embryonic development had no effect. The transfer of an 8-cell embryo with a 16-cell embryo led to the birth of heterosexual twins, indicating that the uterine milieu of Day 5 to 6 recipients may be tolerated by the out-of-phase 8-cell embryo, at least in the presence of a more mature embryo. Embryo quality had the greatest effect on pregnancy rate as it was higher (P < 0.005) after the transfer of Grade I than Grade III embryos (6/10, 60.0% vs 3/36, 13.9%). Assessment of returns to estrus indicated that among nonpregnant recipients, 17/67 (25.4%) embryos never matured sufficiently to prevent luteolysis through maternal recognition of pregnancy (MRP), while 14/67 (20.8%) embryos probably died following MRP. These results indicate that efforts to increase pregnancy rate following embryo transfer in buffalo should include prevention of luteolysis during the first week of transfer and a reduction in the incidence of embryonic mortality.     

Embryo survival in pseudopregnant and in pregnant but genetically semi-sterile recipients after nonsurgical embryo transfer in the mouse

A new nonsurgical embryo transfer technique was used in the mouse that yielded survival rates of between 40 and 70% depending on embryo stage and, possibly, on the degree of synchrony between the embryo and recipient. Three variables were tested using this embryo transfer technique: a) pseudopregnant recipients vs pregnant but genetically semi-sterile recipients, b) embryos resulting from superovulation vs embryos from natural ovulation, and c) 12-hour vs 24-hour asynchrony between donors and recipients. None of these variables significantly affected the pregnancy rate or the percentage of transferred embryos developing to term. The pregnancy rates were between 77 and 90% in 6 experimental groups of 8 to 13 females. Survival rates were between 41 and 63% when all recipients were considered and between 53 and 68% when only the pregnant recipients were included. The embryo transfer procedure influenced litter size composition of the endogenous conceptuses of the semi-sterile recipients. Too many females were devoid of these. Recipients of expanded blastocysts had significantly better transfer results than recipients that also received morulae and early blastocysts. It was concluded that the transfer success rates were influenced by the recipients and possibly by their preparation for transfer.     

Embryonic and calving losses in bovine mixed-breed twins induced by transfer of in vitro-produced embryos to bred recipients

One or two in vitro-produced (IVP) Japanese Black (JB) cattle embryos at 8 days after in vitro fertilization were transferred to the contralateral uterine horn of previously bred Japanese Shorthorn (JSH) or JSH-JB cross recipients, and then the occurrence of early embryonic death, abortion during mid- and late gestation, and calving loss were recorded. The survival rate of embryos, including indigenous ones, was not affected by the number of embryos transferred, and a significantly higher twinning rate (68% of pregnant recipients at 80 days after transfer) was achieved when two IVP embryos were transferred, as compared with the rate when one IVP embryo was transferred (24%). In late ET (recipients at 8.5-9.0 days after the onset of oestrus), the embryo survival rate (22%) and the pregnancy rate (42%) at 80 days after ET were significantly lower than those rates in the synchronous ET (recipients at 8.0 days after the onset of oestrus; 47 and 79%, respectively). In the early ET (recipients at 6.0-7.5 days after the onset of oestrus), no significant differences from the synchronous ET were detected in these rates. Twenty-six percent of twin pregnant recipients were aborted during mid- or late-pregnancy, and 39% of twin calves were stillborn. The mean gestation length of the twin-bearing JSH dams (276 days) was 1 week shorter than that of the single-bearing JSH dams, and it was 2 weeks shorter than that of the JB dams bearing a single JB calf derived from the IVP embryos. The longer gestation length of single JB calves derived from IVP embryos resulted in a significantly higher mean birth weight than that of in vivo control calves with the standard length of gestation. In conclusion, the number of embryos to be transferred did not affect the embryo survival rate, and the transfer of two IVP embryos to previously inseminated recipients induced a significantly higher twinning rate during early pregnancy than that of one IVP embryo transfer. The incidence of embryonic losses during early pregnancy increased when Day 8 embryos were transferred to the recipients later in the oestrous cycle (>8.0 days). The results suggested that one cause of the high rate of abortions and stillbirths in twin-bearing dams is the difference in the mean gestation length between the native JSH and JB foetuses derived from transferred IVP embryos.     

Collection and transfer of multiple embryos in the mare

A pituitary extract was used to induce multiple ovulations in mares to determine whether day-7 embryos from multiple ovulators were viable as indicated by their ability to develop when transferred to recipients. There were more ovulations/donor for induced multiple-ovulating mares than for control single-ovulating mares (4.6 +/- 0.5 vs 1.0 +/- 0.0; n=14). The embryo collection rate per ovulation was similar for multiple ovulators (0.6 +/- 0.1 embryos/ovulation) and single ovulators (0.7 +/- 0.1). The embryo collection rate per donor, therefore, was higher (P<0.01) for the multiple ovulators (2.9 +/- 0.7 vs 0.7 +/- 0.1). The transfer success rate per embryo at day 21 was different (P<0.05) among recipients which received an embryo from control single-ovulating donors (7 8 ), multiple ovulators from which a single embryo was recovered (2 2 ), and multiple ovulators from which multiple embryos were recovered (9 19 ). The recipient pregnancy rate/donor at day 21 was 88% (7 8 ) for single-ovulating controls and 138% (11 8 ) for induced multiple ovulators. Results indicate that the survivability of day-7 embryos from multiple-ovulating donors was reduced. However, despite the reduced survival rate/embryo, the number of pregnant recipients/donor was increased by induction of multiple ovulations because of the increased number of embryos available for transfer.     

Early pregnancy diagnosis by palpation per rectum: influence on embryo/fetal viability in dairy cattle

The objective was to estimate the effect of palpation per rectum (for early pregnancy diagnosis) on embryo/fetal viability in dairy cattle. A controlled, randomized block-design experiment with two blocks, one by category, and the other by number of embryos, was conducted. Five-hundred-and-twenty pregnant dairy cows and heifers with a viable embryo detected by transrectal ultrasonography (TRUS) between days 29 and 32 after AI were included. The pregnant females were randomly allocated into two nearly equal groups: palpation per rectum (PAL group; n=258) and no palpation per rectum (NPAL group; n=262). The PAL group was submitted to palpation per rectum (PPR) using the fetal membrane slip (FMS) technique once between days 34 and 41 of pregnancy. The fetal membrane slip consisted of compressing the pregnant uterine horn and allowing the chorioallantoic membrane to slip between the fingers. Both groups were submitted to two additional TRUS at days 45 and 60 of pregnancy, to monitor the potential immediate and delayed deleterious effects of PPR on embryo and fetal viability, respectively. A diagnosis of embryo/fetal death was made when there was no embryo/fetal heart beat or the absence of positive signs of pregnancy in an animal previously diagnosed pregnant, or the presence of signs of embryo/fetal degeneration. The overall rate of embryo/fetal death was 14.0% (73/520). Embryonic death (10%; 52/520) was higher than fetal death (4.5%; 21/468; P<0.001). Embryo/fetal mortality was higher in cows (16.4%; 59/360) than in heifers (8.8%; 14/160; P<0.025) and in cattle with twin (25.5%; 12/47) versus singleton pregnancies (12.9%; 61/473; P<0.025), but was not different (P>0.05) between PAL (14.7%; 38/258) and NPAL (13.4%; 35/262). In conclusion, PPR between days 34 and 41 of pregnancy using the fetal membrane slip technique did not affect embryo/fetal viability.     

Evaluating recipient and embryo factors that affect pregnancy rates of embryo transfer in beef cattle

The objectives of this experiment were to determine the effects of corpus luteum characteristics, progesterone concentration, donor-recipient synchrony, embryo quality, type, and developmental stage on pregnancy rates after embryo transfer. We synchronized 763 potential recipients for estrus using one of two synchronization protocols: two doses of PGF2alpha (25 mg i.m.) given 11 d apart (Location 1); and, a single norgestomet implant for 7 d with one dose of PGF2alpha (25 mg i.m.) 24 h before implant removal (Location 2). At embryo transfer, ovaries were examined by rectal palpation and ultrasonography. Of the 526 recipients presented for embryo transfer, 122 received a fresh embryo and 326 received a frozen embryo. Pregnancy rates were greater (P < 0.05) with fresh embryos (83%) than frozen-thawed embryos (69%). Pregnancy rates were not affected by embryo grade, embryo stage, donor-recipient synchrony, or the palpated integrity of the CL. Corpus luteum diameter and luteal tissue volume increased as days post-estrus for the recipients increased. However, pregnancy rates did not differ among recipients receiving embryos 6.5 to 8.5 days after estrus (P > 0.1). There was a significant, positive simple correlation between CL diameter or luteal tissue volume and plasma progesterone concentration (r = 0.15, P < 0.01 and r = 0.18, P < 0.01, respectively). There were no significant differences in mean CL diameter, luteal volume or plasma progesterone concentration among recipients that did or did not become pregnant after embryo transfer. We conclude that suitability of a potential embryo transfer recipient is determined by observed estrus and a palpable corpus luteum, regardless of size or quality.     

Pregnancy rates and births after unilateral or bilateral transfer of bovine embryos produced in vitro.

Late morulae and blastocysts produced in vitro were nonsurgically transferred to heifers by unilateral (n = 184) or bilateral (n = 94) transfer. Of the recipients, 58% had serum progesterone values greater than 1.4 ng ml-1 on day 21 and rectal palpation on day 35 showed that 50% (138 of 278) were pregnant. The embryonic mortality rate between days 21 and 35 was estimated to be about 14% and between days 36 and 90 to be about 12%. Of the animals, 8% aborted between days 91 and 250 of pregnancy. No difference was observed in pregnancy rates between unilateral transfer of one (47%) or two embryos (49%) and bilateral transfer (53%), or in the twinning rate between bilateral transfer (42%) and unilateral transfer of two embryos (33%). The pregnancy rate was 54% with embryos evaluated as morphologically excellent or good, 51% with fair embryos and 26% with poor ones. A higher pregnancy rate (60%) was obtained after embryo transfer when the synchrony between recipient and embryo was -1 day.     

Effect of asynchronous transfer and oestrogen administration on survival and development of porcine embryos.

Results indicate that recovery of embryos on Days 11 and 13 of pregnancy was reduced for Day 5 embryos transferred to recipients on Day 6 of their oestrous cycle and was greatly reduced when embryos were transferred to recipients on Day 7 of the cycle (P less than 0.01). Administration of oestradiol-17 beta on Day 11 of the recipient's cycle did not appear to affect embryo development on Day 13. Day 6 embryos transferred to recipients on Day 8 of the oestrous cycle deteriorated rapidly within 24 h of transfer; there was no recovery of embryos from the uterus after 36 h. Treatment of pregnant gilts with 1 mg oestradiol-17 beta (i.v.) on Day 10.5 resulted in total embryonic loss by Day 23, but pregnancy rates of gilts treated with oestradiol-17 beta on Day 12 were similar to those of vehicle-treated gilts (60.6 vs. 71.4%).     

Transfer of cow blastocysts after sexing

We obtained 174 Charolais cow blastocysts at day 13 (D(13)) after superovulation and slaughter of 24 donor heifers. Using de Wecker's scissors, we sampled 1 10 to 1 3 of the total blastocyst volume. After colcemid treatment, hypotonic shock, fixation and Giemsa staining, the sex of 149 blastocysts (60 %) was determined. The main difficulty in sexing was the absence of metaphase or the presence of incomplete metaphases on the preparations. During manipulation, the portion of the blastocyst to be transferred was stored at 37 degrees C in a medium of 20 % fetal calf serum containing 80 % B(2) under an atmosphere of 90 % nitrogen, 5 % O(2) and 5 % CO(2) for 6 to 7 hours. After sex determination, the blastocysts were put into an artificial insemination straw and placed with a catheter at the base of the uterine horns. Twenty-eight blastocysts were transferred into both horns of 14 recipient Charolais heifers; 4 heifers (28 %) had an extended cycle and 3 (21 %) were pregnant. When 2, 3 or 4 blastocysts were transferred into the ipsilateral horn with the corpus luteum, 20 to 25 % of the recipients had an extended cycle and 37, 40 and 75 %, respectively, were pregnant. The number of surviving embryos per recipient never exceeded 2.     

Successful superovulation, nonsurgical collection and transfer of embryos from Brahman cows

Nonsurgical recoveries and transfers of embryos were performed at the McKellar Embryo Transplant Center from 122 superovulated Brahman cows. FSH-P (Armour) was used to superovulate all cows at dose levels ranging from 36 to 48 mg total FSH-P. Luteal regression was induced by use of 40 mg PGF(2(alpha)) in all 122 cows. Embryos were transferred into recipients 6, 7 or 8 days after observed estrus. Embryos were successfully collected from 82% of the FSH-P treated cows. The dose level of FSH-P affected numbers of embryos collected (P<.05). Numbers of embryos collected from cows superovulated with 36-38, 40, 42, 43, 44, 45, 46 and 47-48 mg FSH-P were 2.8 +/- 1.0, 6.8 +/- 1.1, 9.4 +/- 1.4, 10.0 +/- 2.7, 7.1 +/- 1.6, 6.8 +/- 2.0, 5.0 +/- 1.7 and 4.6 +/- 2.0 embryos, respectively. The dose level of FSH-P also affected numbers of embryos transferred (P<.10). Number of embryos transferred from cows superovulated with 36-38, 40, 42, 43, 44, 45, 46 and 47-48 mg FSH-P were 2.8 +/- 1.9, 5.2 +/- 0.9, 6.9 +/- 1.2, 6.7 +/- 2.1, 4.8 +/- 1.3, 5.1 +/- 1.4, 3.4 +/- 1.2 and 3.2 +/- 2.1 embryos, respectively. The developmental stage (D) of the embryo was also a factor in pregnancy rate of recipients (morula = 13.8%, blastocyst = 22.1% and expanded blastocyst = 29.9%; P<.005). The skill of the technician (T) transferring the embryo had a dramatic effect upon subsequent pregnancy rate of the recipients (T 1 = 46.0% vs T 2 = 22.6% pregnancy rate; P<.005). Pregnancy rate of recipients was also affected by the stage postestrus (S) at which the embryo was transferred (day 6 = 23.5%, day 7 = 25.5% and day 8 = 42.3% pregnancy rate; P<.05). Interactions were found between T x S, T x D, S x D and T x S x D (P<.05). These data indicate that use of 40, 42, or 43 mg total doses of FSH-P were quite effective in superovulating the Brahman cow. Recipients transferred on day 8 postestrus achieved higher pregnancy rates than recipients transferred on days 6 or 7 postestrus. Embryos transferred in the expanded blastocyst stage of development proved to yield the highest pregnancy rates in recipients.     

The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus

Embryos were collected at the 4-10-cell stage from the oviducts (Day 4; Day 1 = ovulation) or as morulae (Day 7) from the uterus of marmosets and frozen in 1.5 M-DMSO (Days 4 and 7) or 1.0 M-glycerol (Day 4 only), using a slow freezing and thawing technique. Of 22 Day-4 embryos frozen in DMSO, 18 were recovered and 16 of these were transferred to 10 synchronized recipients; 7 recipients became pregnant compared with all 7 control recipients receiving 10 unfrozen embryos. Fifteen frozen-thawed morulae were transferred to 9 Day-6 recipients; the pregnancy rate (55.6%) was lower than for control embryos (85.7%). Embryos frozen in glycerol suffered severe osmotic stress during glycerol addition and removal. Of 8 recipients, 3 (37.5%) became pregnant but only one fetus was carried to term. These results on embryo collection, freezing and transfer in the marmoset have important implications for developing improved methods for freezing human embryos and the breeding of endangered primates.     

Pregnancy rates with bisected bovine embryos

A method for producing identical twin calves is described in which 5.5 to 7.5 day bovine embryos were bisected using a fine microsurgical blade. The resulting demi-embryos were transferred surgically or nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/- 2 days), one demi-embryo per recipient. The mean pregnancy rate per demi-embryo was 48% (157/330), and per whole embryo split, it was 95% (157/165). Pregnancy rate was dependent on the stage of embryonic development. The pregnancy rate from bisected early morulae, 16% (7/44), was lower than that from bisected early blastocysts, 60% (58/96) P<0.01. The rate of identical twin production per embryo split reflected these pregnancy rates. Bisected blastocysts resulted in more identical twin sets, 39% (24/62), than early morulae, 9% (2/22) P<0.01. When used with normal embryo transfer procedures, this technique provides a method for routine production of identical twin calves. Furthermore, more calves are produced per two demi-embryos than per whole embryo.     

Transfer of vitrified blastocysts from one or two superovulated Large White Hyperprolific donors to Meishan recipients: reproductive parameters at Day 30 of pregnancy

The present study was designed to determine the effect of pooling embryos from two donors on the reproductive success of transfer of vitrified/warmed porcine blastocysts. Intact blastocysts were collected from superovulated Large White Hyperprolific gilts (n = 24) on Days 5-5.5 after artificial insemination. Embryos were recovered by flushing the uterine horns, and unhatched blastocysts were selected. Vitrification and warming were performed as described by Berthelot et al. [Cryobiology 41(2000) 116]. To evaluate in vitro development, 37 vitrified/warmed blastocysts were cultured, non-vitrified embryos (n = 48) were used as controls. Embryo transfers were conducted in asynchronous (-24 h) Meishan gilts (n = 20). Twenty vitrified/warmed blastocysts were surgically transferred into one uterine horn. Ten recipients received embryos from one donor (Group 1) and the other 10 transfers were performed with mixed embryos from two donors (Group 2). Pregnancy was assessed ultrasonographically at Day 25 after estrus and recipients were slaughtered at Day 30 after transfer. In vitro survival rate of the vitrified/warmed blastocysts was lower (P < 0.01) than that from control embryos (73.0% versus 93.7%). The pregnancy rate for Group 1 (70%) was not different (P > 0.05) than that from Group 2 (90%). No significant differences were detected between Groups 1 and 2 for in vivo embryo development (number fetuses/transferred embryos in pregnant recipients) or in vivo embryo survival (number viable fetuses/transferred embryos in pregnant recipients). However, the in vivo efficiency (number viable fetuses/total transferred embryos) was higher (P < 0.05) when transfers were performed with embryos from two donors (19.5% versus 30.5%). These results indicate that pooling embryos from two donors increases the in vivo efficiency after transfer of vitrified/warmed porcine blastocysts.     

Invitro fertilization of cow ova

Embryos were recovered from superovulated heifers and transferred non-surgically through the cervix of heifers via an ureteral catheter. The ureteral catheter was passed into the anterior portion of the uterine horn ipsilateral to the corpus luteum through a series of concentric stainless steel tubes. One morula or blastocyst was transferred to each of 95 heifers.Eighteen heifers (19%) were pregnant 55 to 60 days later. The pregnancy rate was not significantly influenced by treatment of the vagina with antibiotics before transfer; whether a morula or blastocyst was transferred; or whether ovulation occurred from the left or right ovary. There were no significant differences among the three technicians. Fifty of the 77 recipients that were not pregnant at 60 days of gestation were observed in estrus after 18–23.5 or 37–44.5 days, representing normal or double the normal estrous cycle lengths. However, 11 showed a shortened return to estrus (9–26 days), and 16 had a delayed return to estrus (25–33 or 48–55 days). We conclude that this technique results in premature luteolysis in some recipients and probably in more early embryonic death than normal.