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.     

Splitting of porcine embryos at the four-cell or morula stage

Porcine embryos obtained from estrus-induced prepuberal gilts were split at the 4-cell and morula stage. The in vitro development of the demi-embryos was compared with that of intact control embryos. The intact control embryos developed according to expectation. The in vitro survival of the demi-embryos was inferior to that of the respective controls. The splitting of 4-cell embryos was easier to accomplish and more efficient than the splitting of morulae. The in vitro development of the 4-cell embryos was also slightly better, although this difference was not significant. In vitro development of demi-embryos originating from morulae split at room temperature was slightly although nonsignificantly inferior to that of demi-embryos from morulae split on a warming stage at 37 degrees C. Between 14 and 18 demi-embryos were transferred to synchronous recipient gilts after 24 to 48 h of culture. Of 3 gilts receiving split 4-cell embryos, 1 gilt aborted 2 normal fetuses on Day 90 of pregnancy and 1 carried a single piglet to term. Of the 4 gilts receiving split morulae, 1 gilt had 4 normal and 3 degenerated fetuses upon slaughter on Day 35 of pregnancy.     

A procedure for successful nonsurgical embryo transfer in swine

The current study was undertaken to develop a successful procedure for the nonsurgical transfer of pig embryos. A total of 663 embryos were surgically collected on Day 4 or 5 from 55 donors, of which 542 embryos of acceptable quality were nonsurgically transferred to 46 recipients. Nonsurgical recipient gilts were sedated 15 min prior to transfer with 20 mg im acepromazine maleate. A disposable insemination spirette with an attached 3-way stopcock was manipulated into the cervix of each gilt. Embryos were expelled from a tomcat catheter into the spirette, and 10 to 12 ml of Whitten's medium were used to flush embryos through the spirette into the reproductive tract. Sixteen (34.8%) recipient gilts did not return to estrus before Day 36, and 10 (21.7%) gilts farrowed with an average litter size of 4.3 +/- 0.7. Embryos were collected from an additional 20 donors and were surgically transferred to an additional 19 recipients. Surgical transfers conducted at the same time as the nonsurgical transfers resulted in 12 (63.2%) gilts farrowing and 7.1 +/- 0.6 pigs were born per litter. In conclusion, a procedure has been developed for nonsurgical transfer of swine embryos which simplifies the process of embryo transfer and which may increase the potential for utilization of embryo transfer technologies by swine producers.     

Conceptus development in large white and prolific Chinese Meishan pigs

Large White (LW) and Meishan (MS) gilts were killed on Days 8, 10, 11, 12, 14 and 30 of gestation. Mean diameters (mm) for MS and LW conceptuses, respectively, were: Day 8, 0.45 and 0.69; Day 10, 2.7 and 1.9; Day 11, 5.3 and 2.7, with the differences among days being affected by breed (P less than 0.01). Variation in diameter among conceptuses from LW gilts was greater (P less than 0.01) than that for MS gilts on Days 8-11, respectively: Day 8, 20 and 46%; Day 10, 29 and 38%; and Day 11, 22 and 44%. Conceptuses had elongated in 3 of 5 MS and 1 of 4 LW gilts on Day 11, 6 and 6 MS and 2 of 4 LW gilts on Day 12 and all gilts of both breeds on Day 14. These results indicate that conceptuses of MS gilts develop more rapidly and more uniformly between Days 8 and 14 of gestation. Overall, embryonic survival for Days 8-12 for gilts not having elongated conceptuses was 90.2% for MS and 73.2% for LW gilts (P less than 0.01). On Day 30 of gestation, embryonic survival was also higher (P less than 0.01) for MS (89%) than LW (55%) gilts. However, embryonic weight, crown-rump length, placental length, allantoic fluid volume, amniotic fluid volume, as well as total glucose, fructose and protein in allantoic fluid were not affected by breed. Placental weight was greater (P less than 0.01) for LW gilts. Uterine development at Day 30 of gestation, based on total length and weight of uterine horns, width of uterine horns, total endometrial surface area and total endometrial weight was greater (P less than 0.01) for LW gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Farrowing rates and litter size following transfer of vitrified porcine embryos into a commercial swine herd

The objective was to determine farrowing rates and litter sizes that could be achieved in a typical farm-to-farm porcine embryo transfer program using vitrified blastocysts that were zona pellucida intact when cryopreserved. The embryos were transferred surgically on-farm into recipient sows that were managed throughout gestation and farrowing under the same conditions as other sows in the herd. Twenty recipient sows (mean parity 2.1) received a total of 568 embryos; seven received 203 embryos derived from donor sows, five received 139 embryos from gilts and eight received a mixture of 161 embryos from sows and 65 from gilts. Sixteen sows (80%) were confirmed pregnant at approximately 35 days gestation, 15 farrowed at full term (farrowing rate 75%). One sow died during gestation (with a total of 18 fetuses in utero). A total of 123 piglets were born (mean, 8.2), of which 115 were born alive (mean, 7.7). Of the 568 embryos transferred to all 20 sows, 21.6% resulted in piglets born and 29.0% survived to produce piglets in sows that farrowed. There were no significant differences in embryo survival among sow, gilt or mixed sow and gilt embryos. The ratio of males to females was 71/52 and the mean birth weight was 1.6 kg (range 0.6-2.6 kg). In conclusion, vitrified zona pellucida intact embryos can be used to transfer genetic material from farm-to-farm with acceptable reproductive performance.     

Effect of hCG administration at the onset of oestrus on early embryo survival and development in Meishan gilts

A study was designed to advance the time of the ovulatory luteinizing hormone (LH) surge in Meishan gilts by human chorionic gonadotrophin (hCG) administration at the onset of oestrus and assess the effect on embryo survival and development. Twelve Meishan gilts were observed six times daily for oestrous behaviour and bred at 24, 36 and 48 h after observed oestrous onset. Six of those gilts were administered an ovulatory dose of hCG (500 IU) at observed oestrous onset. Blood samples were collected at oestrous onset (Day —2) and on Days 0, 2, 6, 9, 13, 16, 20, 23, 27 and 30 of gestation. All gilts were slaughtered on Day 30 of gestation and embryo survival and conceptus development assessed. Ovulation rate did not differ between control and hCG treated gilts (18.5 and 17.7 respectively; P>0.1) while the number of live conceptuses per gilt (17.2 and 12.8 respectively; P<0.08) and embryo survival rate (92.1 and 75.8% respectively; P<0.1) both tended to be reduced by the hCG treatment. Placental weight (17.2 and 23.1 g; P<0.01) was significantly increased in hCG treated gilts, while embryo weight (1.2 and 1.4 g; P<0.06) and placental length (42.8 and 47.2 cm; P<0.07) both tended to be increased in hCG treated gilts. Crown rump length (P>0.1) and allantoic fluid volume (P>0.1) did not differ between the treatment groups. Serum progesterone concentrations did not differ with treatment overall (P>0.1) but were significantly elevated (P< 0.05) at 48 h postoestrus in the hCG treated gilts compared to control gilts. Overall, these results indicate that advancing the time of the LH surge to oestrous onset, as in European breeds, compromised embryo survival and suggests that the longer time interval between oestrous onset and ovulation is important for the high rate of embryo survival in the Meishan pig.     

Effect of asynchronous superinduction on embryo survival and range of blastocyst development in swine

The importance of uniform development of blastocysts was examined by comparing the effects of asynchronous superinduction (Day 6 embryos into Day 7 pregnant recipients and Day 7 embryos into Day 6 pregnant recipients) on the range of embryo development at Days 12 and 13 to subsequent survival to Day 30. Twenty gilts were used to produce five Day 7 recipients that received Day 6 embryos and five Day 6 recipients that received Day 7 embryos. Embryos from the Day 7 and Day 6 recipients were examined 6 days later. Recovered embryos ranged morphologically from spherical to filamentous blastocysts. This range of embryos was within the limits of that previously observed for naturally mated sows. However, recovered blastocysts from the Day 6 embryos transferred into Day 7 recipients were morphologically more variable and proportionately less developed than the blastocysts from the Day 7 embryos transferred into Day 6 recipients. Forty additional gilts were subsequently utilized to generate 20 recipients (10 recipients per transfer group) that were examined on Day 30. More Day 7 embryos transferred into Day 6 recipients survived (p less than 0.05) than Day 6 embryos transferred into Day 7 recipients. These experiments suggested that greater variation in early development of embryos, within litters, subsequently resulted in greater mortality of embryos.     

Effect of variation in embryo stage on the establishment of pregnancy, and embryo survival and growth in ewes with two embryos

Embryos at different stages of development were transferred to recipient ewes on Day 6 to investigate the effect of variation in stage of development on embryo survival and growth. Three groups of ewes received 2 embryos that were at the same stage of development, Day 4, Day 6 or Day 8. A fourth group received 1 Day-4 and 1 Day-8 embryo. At autopsy on recipient Day 34 there were no significant differences in embryo survival (Day 4, 34%; Day 6, 50%; Day 8, 46%; and Day 4 and 8, 48%). Fetuses developing from Day-8 embryos were heavier than others (Day 4, 1.10 +/- 0.06 g; Day 6, 1.15 +/- 0.06 g; Day 8, 1.41 +/- 0.08 g; P less than 0.05). In Group 4 neither survival nor growth of embryos was significantly affected by the presence of an embryo at a different stage of development. The ability of the uterus to stimulate development of a relatively retarded embryo is confirmed. Apparently the uterus has less effect in slowing the development of advanced embryos.     

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%).     

Age at puberty, ovulation rate, uterine length, prenatal survival and litter size in Chinese Meishan and Yorkshire females

Studies on the ovulation rate, prenatal survival and litter size of Chinese Meishan pigs have given widely divergent results depending on the extent of inbreeding of the animals, their original genetic diversity, the age and parity, and the conditions of management. To obtain meaningful results, it is necessary to characterize the population under study. The following report characterizes populations of Meishan and Yorkshire of a widely diverse background. First farrowing data were collected on 21 Meishan and 20 Yorkshire gilts. Meishan gilts had 12.4 fully formed piglets and Yorkshire gilts had 7.4 fully formed piglets (P < 0.01). Meishan gilts averaged 1.86 mummified fetuses per litter vs 0.05 per Yorkshire litter (P < 0.01). Yorkshire piglets averaged 1.3 kg body weight at birth vs 0.9 kg for Meishan piglets (P < 0.01). At 47 days of second gestation, 19 Meishan and 12 Yorkshire sows averaged 22.7 and 16.3 corpora lutea (CL), respectively (P < 0.01). Uterine length and number of fetuses were not different (P > 0.40) in the two breeds. Daily estrous detection of 50 Meishan and 34 Yorkshire gilts began at 60 and 120 days of age, respectively. Meishan gilts reached sexual maturity at 95 days of age, which was 105 days earlier than Yorkshire gilts (P < 0.01). Meishan gilts were in estrus nearly 1 day longer than Yorkshire gilts at first, second and third estrus (P < 0.05). No differences in cycle length between breeds were detected for the first or second estrous cycle (P > 0.60). Nineteen Meishan gilts were slaughtered at 51 days of gestation and their reproductive tracts were recovered. The mean number of dissected CL (17.0), number of fetuses (13.1), total uterine length (396 cm), spacing per fetus (29.9 cm), allantoic (124.9 ml) and amniotic (32.2 ml) volumes, crown-rump length (82.8 mm), weight (35.4 g), sex, and direction of each fetus were determined. Chinese Meishan gilts reached puberty much earlier and were in estrus longer than Yorkshire gilts and Meishan sows had more CL than Yorkshire sows.     

In vitro development of embryos from superovulated gilts treated with the progesterone agonist, altrenogest (Regu-Mate) or the prostaglandin analogue, cloprostenol (Planate)

This study was conducted to compare the in vitro development of embryos from superovulated postpubertal gilts synchronized with progesterone agonist altrenogest (REG, Regu-Mate) and those from superovulated prepubertal gilts synchronized with prostaglandin analogue cloprostenol (PLA, Planate). Ten postpubertal gilts that had exhibited estrus at least once were fed 20 mg/d of REG from Day 0 (the first day of treatment, may have been any day of the estrous cycle) to Day 17. The gilts received intramuscularly (im) 1500 IU of equine chorionic gonadotropin (eCG) on the afternoon of Day 17, followed by 1000 IU of human chorionic gonadotropin (hCG) 84 h later. Eight prepubertal gilts received intramuscularly one dose of a combination of 400 IU of eCG and 200 IU of hCG (PG 600) on Day 0 (the first day of treatment), followed by 750 IU of hCG on Day 3. From Day 16 to Day 19, the prepubertal gilts received 350 mg/d of PLA, followed by 1500 IU of eCG on the afternoon of Day 19, then 1000 IU of hCG 84 h later. Gilts were checked for estrus with an intact boar. At estrus, all gilts were artificially inseminated and/or mated twice at 12-h intervals. Then 50 to 54 h after the hCG injection, a mid-ventral laparotomy was performed on each gilt. Corpora albicans (CA) and corpora hemorrhagica (CH) were counted, and oviducts were flushed in situ. The embryos recovered (1- to 2-cell) were cultured in modified Whitten's medium at 38.5 degrees C under an atmosphere of 5% CO2 in air for 144 h. The number of CA per gilt did not differ between the postpubertal and prepubertal gilts (11.9 vs 7.9, respectively; P > 0.05). However, the number of CH per gilt (27.5 vs 18.1, P = 0.05) and the number of embryos per gilt (26.2 vs 15.3, P < 0.05) were higher in postpubertal gilts than in prepubertal gilts. Furthermore, after 144 h of in vitro culture, the percentage of embryos cleaving to the >-16-cell (morula + blastocysts) or > or =32-cell (blastocysts) was greater (P < 0.05) in prepubertal gilts than in postpubertal gilts (85.2 vs 68.5, 55.7 vs 44.2, respectively). The total numbers of embryos examined were 122 and 260 in prepubertal and postpubertal gilts, respectively. These results show that postpubertal gilts treated with REG produced a higher number of embryos. However, better embryo development was noted with zygotes from prepubertal gilts primed with exogenous gonadotrophin, followed by synchronization with prostaglandin before induction of superovulation and insemination.     

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to −6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), −1 (68%), −2 (63%), −3 (66%), −4 (76%), −5 (61%), and −6 (27%). The −6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to −5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.     

Antagonists of embryo-derived platelet-activating factor act by inhibiting the ability of the mouse embryo to implant.

This study utilized the transfer of preimplantation embryos to pseudo-pregnant mice to determine whether PAF-antagonists act primarily on the maternal or embryonic components of implantation. The first experiment used reciprocal embryo transfers, in which blastocysts from mice treated with PAF antagonist (SRI 63-441) or saline (controls), from Days 1 to 4 of pregnancy, were transferred to Day-3 pseudo-pregnant recipients which were also treated with SRI 63-441 or saline on Days 1-4 of pregnancy. The antagonist (40 micrograms) was administered at 16:00 h on Day 1 and at 09:00 h on Days 2-4 of pregnancy. The percentage of the transferred embryos which implanted was determined on Day 8 of pregnancy. Treatment of the recipient or the donor female with SRI 63-441 resulted in a reduction in implantation rate, from a control level of 45% to 33.8% or 34.7% (P less than 0.0002, P less than 0.007) respectively. These results suggest that the PAF antagonist affected implantation at the embryonic and maternal levels. However, when the blastocysts were transferred to Day-4 pseudopregnant recipients, treatment of the donor female had a dramatic effect on the implantation rate, resulting in a reduction of 64% (from 40% to 14.3%, P less than 0.04), while treatment of the recipient female had no significant effect. In this later experiment the transferred embryos were exposed to the recipient uterine environment for a shorter period before implantation. These results suggest that PAF antagonists affected implantation at the embryonic level and did not adversely affect maternal physiology.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sexual maturation and morphological development of the reproductive tract in large white and prolific Chinese Meishan pigs

Body weight of Large White gilts was greater at birth, weaning, 5 months of age and at slaughter; however, Meishan gilts reached puberty at an earlier age (91 +/- 2 vs 192 +/- 3 days, P less than 0.01), had longer periods of oestrus (60 +/- 2 vs 49 +/- 2 h, P less than 0.01) and experienced more oestrous cycles (7 +/- 0.4 vs 4 +/- 0.4, P less than 0.01) before slaughter. The interoestrous interval was longer (P less than 0.01) for Large White gilts (19.8 +/- 0.2 vs 19.1 +/- 0.2 days). At slaughter, uterine length (P less than 0.05), uterine weight, width of uterine horns, endometrial surface area, endometrial weight and percentage of uterine weight represented by endometrium was greater (P less than 0.01) for Large White gilts. However, breed differences were not significant when slaughter weight was included in analyses as a covariate. This indicated that development of the reproductive tract was proportionate to body weight at slaughter for each breed. When body weight at slaughter was included as a covariate, effects of day of the oestrous cycle and pregnancy on uterine width, uterine weight, endometrial surface area and endometrial weight were detected (P less than 0.01) and for uterine length there was a day-by-status interaction (P less than 0.01). Total number of CL (P less than 0.05) and total ovarian weight (P less than 0.05) were also greater for Large White gilts independent of body weight at slaughter. There were more CL in left ovaries for Meishan (8.1 +/- 0.4 vs 6.6 +/- 0.4) and Large White (8.4 +/- 0.4 vs 7.9 +/- 0.5) gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphology and morphometry of in vivo- and in vitro-produced bovine concepti from early pregnancy to term and association with high birth weights

This study was designed to characterize conceptus development based on pre- and postnatal measurements of in vivo- and in vitro-derived bovine pregnancies. In vivo-produced embryos were obtained after superovulation, whereas in vitro-produced embryos were derived from established procedures for bovine IVM, IVF and IVC. Blastocysts were transferred to recipients to obtain pregnancies of single (in vivo/singleton or in vitro/singleton groups) or twin fetuses (in vitro/twins group). Ultrasonographic examinations were performed weekly, from Day 30 of gestation through term. Videotaped images were digitized, and still-frames were used for the measurement of conceptus traits. Calves and fetal membranes (FM) were examined and measured upon delivery. In vitro-produced fetuses were smaller than in vivo controls (P < 0.05) during early pregnancy (Day 37 to Day 58), but in vitro/singletons presented significantly higher weights at birth than in vivo/control and in vitro/twin calves (P < 0.05). From late first trimester of pregnancy (Day 72 to Day 93), placentomes surrounding in vitro-derived singleton fetuses were longer and thinner than controls (P < 0.05). At term, the presence of giant cotyledons in the fetal membranes in the in vitro group was associated with a larger cotyledonary surface area in the fetal horn (P < 0.05). The biphasic growth pattern seen in in vitro-produced pregnancies was characterized by conceptus growth retardation during early pregnancy, followed by changes in the development of the placental tissue. Resulting high birth weights may be a consequence of aberrant placental development due to the disruption of the placental restraint on fetal growth toward the end of pregnancy.     

Cleavage of pig embryos after labeling with fluorescent dyes

In studies on embryonic development, treated and control ova could be co-mixed before transfer to recipients if nontoxic labels for ova were available. These experiments were conducted to determine whether pig ova would continue to cleave after being stained with the fluorochromes tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC). In the first experiment, pig ova stained with TRITC and unstained control ova were transferred into opposite oviducts of recipient gilts. In the second experiment, ova stained with TRITC and ova stained with FITC were transferred into opposite oviducts of recipient gilts. Embryos were recovered 96 h after transfer (Day 6; Day 0 = onset of estrus), the presence of fluorescence was determined, and the number of nuclei per embryo was assessed. Stained ova retained sufficient fluorochrome to permit detection until the zonae pellucidae were shed. Development of embryos stained with TRITC was equal to that of unstained control embryos. However, development of embryos stained with FITC appeared slightly retarded in comparison to that of TRITC-stained embryos. These findings demonstrate the efficacy of the fluorescent staining technique for pig ova during the first six days of pregnancy.     

Survival of equine embryos transferred to normal and subfertile mares

To test the hypothesis that an abnormal uterine environment was a cause of early embryonic loss in subfertile mares, morphologically normal embryos were transferred to normal mares (n = 20) and subfertile mares (n = 20), and embryo survival rates were compared. Embryos were recovered nonsurgically at Days 7 to 8 postovulation and transferred surgically to normal and subfertile mares that had ovulated on the same day or within 2 d after a donor. Survival of transferred embryos was monitored by ultrasonography of the recipient mare's uterus from Day 9 through Day 28 postovulation. There were no significant differences (P > 0.5) in the embryo survival rates at Day 12 (11 20 vs 9 20 ) or Day 28 (10 20 vs 8 20 ) for normal or subfertile mares, respectively. The uterine environment of subfertile mares was apparently adequate to support the development of transferred embryos from Days 7 or 8 through Day 28 postovulation.     

Ovarian response, embryo recovery and results of embryo transfer in a Hungarian native pig breed

The objective of the study was to use embryo transfer (ET) for propagation of the Swallow Belly Mangalica population. Mangalica is a native Hungarian pig breed adapted to extreme climate and housing conditions and distinguished for excellent meat and fat quality. However, due to their weak reproductive characteristics and relatively high fat proportion, Mangalica pigs have been replaced by modern breeds. Now, there is an increased interest again to safeguard the properties of this breed. We conducted two experiments. First, we used a total of 18 puberal Mangalica gilts to determine an optimal superovulatory treatment. Following estrus synchronization with Regumate, we injected gilts with either 750, 1000 or 1250 IU PMSG, followed by 750 IU hCG 80 h later. We scanned ovaries endoscopically 3 days after hCG administration. The application of 1000 and 1250 IU PMSG resulted in a higher rate of ovulation compared to 750 IU (24.2 +/- 3.6 and 21.0 +/- 2.3 vs. 13.7 +/- 2.7 P<0.05). The number of follicular cysts increased after administration of 1250 IU PMSG compared to 750 and 1000 IU (2.0 +/- 1.3 vs. 0.3 +/- 0.7 and 0.2 +/- 0.3, P<0.05). Thus, we chose 1000 IU PMSG for further stimulation of Mangalica gilts. In the second experiment, we induced superovulation in 10 Mangalica donor gilts by 1000 IU PMSG and 750 IU hCG. Gilts were fixed-time inseminated, and then five days later embryo collection was carried out surgically (n=6) or endoscopically (n=4). Out of the 187 ova recovered, 92.5% were at the morula/blastocyst stage. The embryo recovery rate was higher following surgical flushing than following endoscopy (91.5 +/- 4.4% vs. 71.4 +/- 12.7%, P<0.05). Altogether 143 embryos were transferred surgically or endoscopically into 8 Landrace recipients. Surgical and endoscopic transfer of Mangalica embryos into Landrace gilts resulted in pregnancies in 3 and 2 gilts, respectively; thus the overall farrowing rate was 62.5%. The birth of 59 Mangalica piglets from 5 embryo recipients equals an average litter size of 11.8 +/- 1.3, which is two times larger than usual in this breed. Therefore, we concluded that an appropriate inter-breed ET program is a suitable tool to propagate the endangered Mangalica breed.     

Decreased embryonic survival of in-vitro fertilized oocytes in rats is due to retardation of preimplantation development

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2 and allocated to 3 groups. On the evening of Day 0, rats in Groups I and II were allowed to mate. Embryos were collected on Day 4 (Group I, control morulae) or Day 5 (Group II, control blastocysts) and were transferred into the oviduct or uterine horn of Day-4 pregnant recipient rats. On the transfer side of the recipients, the bursa had been peeled from around the ovary to prevent endogenous oocytes from entering the oviduct. For Group III, unmated donors were killed 65-67 h after PMSG injection. Ovulated oocytes recovered from the oviducts were fertilized in vitro and transferred 16-18 h later. Embryos developing from in-vitro fertilized (IVF) oocytes were recovered on Day 5, separated into morulae (Group IIIm) and blastocysts (Group IIIb) and transferred into Day-4 pregnant recipients similar to control embryos. Some embryos from each group were used to determine the mean number of cells/embryo. Embryo recipients were killed on Day 20. After transfer, the development of IVF oocytes was retarded compared to control embryos. IVF morulae contained significantly fewer cells/embryo than did control morulae but were able to implant and grow to fetuses, in proportions similar to controls, if transferred into the oviduct of the recipients. These results suggest that the developmental potential of rat oocytes fertilized in vitro is limited due to asynchrony between the embryo and the uterine environment at the time of implantation, rather than possible defects incurred by the oocyte during the fertilization procedure.