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.     

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.     

Pregnancies following transfer of equine embryos cryopreserved by vitrification

The objective of this study was to investigate the in vitro and in vivo developmental abilities of equine embryos cryopreserved by vitrification. Twenty-eight embryos were recovered from Native pony and Thoroughbred mares at Days 5 to 7 by nonsurgical uterine flushing (detection of ovulation=Day 0). The vitrification solution contained 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in PBS. The embryos were placed for 1 to 2 min in vitrification solution (Group 1) or following exposure to 20% ethylene glycol in PBS for 10 to 20 min (Groups 2 and 3). Single embryos were loaded in 0.25-ml straws, cooled for 1 min in liquid nitrogen vapor and immersed in liquid nitrogen. Straws were warmed in water (20 degrees C, 20 sec), and the contents were expelled with 0.5 M sucrose in PBS. Then the sucrose was diluted in 1-step (Groups 1 and 2) or 4-steps (Group 3). Embryos (n=21) were cultured for 120 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO(2) in air and evaluated morphologically. Development to the hatching or hatched blastocyst stage was obtained in 0 7 , 4 7 and 4 7 embryos in Groups 1, 2 and 3, respectively. An additional 7 embryos were vitrified-warmed according to the treatment of Group 2 (4 embryos) and Group 3 (3 embryos). Five embryos were selected after in vitro culture for 4 h and were transferred nonsurgically into the uterine horn of Day-4 recipient mares. Transfer of 2 embryos (both Day-6 blastocysts: Group-2 treatment) resulted in pregnancies with a viable fetus at Day-60 of the gestation period.     

Studies of FSH-P induced follicular growth in cows

Because cow ovaries do not contain a dominant follicle before Day 3 of the estrous cycle, we hypothesized that gonadotropin treatment early in the estrous cycle would induce growth of multiple follicles and could be used to induce superovulation. In Experiment 1, when 16 cows were treated with FSH-P beginning on Day 2 of the estrous cycle and were slaughtered on Day 5, all cows responded to gonadotropin treatment by exhibiting a large number ( approximately 19) of estrogenactive follicles >/= 6 mm. In Experiment 2, in response to FSH-P treatment from Day 2 to Day 7, and fenprostalene treatment on Day 6, 11 of 15 cows exhibited estrus and had a mean ovulation rate of 23.7 +/- 1.5. In Experiment 3, an FSH-P treatment regimen identical to that used in Experiment 2 was administered to cows beginning either on Day 2 (Day-2 cows; n=14) or Day 10 (Day-10 cows; n=11) of the estrous cycle. Twelve of 14 Day-2 cows and all Day-10 cows exhibited estrus after fenprostalene treatment. Day-2 cows exhibited 34.3 +/- 7.0 ovulations, which was less (P < 0.05) than that exhibited by Day-10 cows (48.3 +/- 4.4). However, the proportion of embryos recovered per corpus luteum was about 2-fold greater (P < 0.05) for Day-2 cows than for Day-10 cows (0.49 +/- 0.08 vs 0.27 +/- 0.06). These data indicate that beginning gonadotropin treatment early in the estrous cycle, when a dominant follicle is not present, provides an efficacious means to induce growth of multiple follicles and superovulation in cows. However, when FSH was administered for 6 d, beginning the treatment on Day 10 also resulted in a consistent and efficacious response.     

Superovulation in heifers given FSH initiated either at day 2 or day 10 of the estrous cycle

The aim of this study was to determine if initiation of superovulation in heifers during the time of development of the first dominant follicle (Days 2 to 6) would give equivalent ovulation and embryo production rates as treatment initiated at mid-cycle. Estrus was synchronized in 60 beef heifers using luprostiol (PG) and they were randomly allocated to treatment with 4.5, 3.5, 2.5 and 1.5 mg of porcine follicle stimulating hormone (FSH) administered twice daily, either on Days 2, 4, 5 and 6 (Day-2 group), respectively, or with similar doses at four consecutive days during mid-cycle (Day-10 group, initiation on Day 9 to 11). All heifers received 500 mug cloprostenol at the fifth FSH injection and 250 mug at the sixth injection. Blood samples for progesterone determination were collected at the time of FSH injections. Heifers were slaughtered 7 d post estrus, and the number of ovulations and large follicles (>/=10mm) were determined on visual inspection of the ovary. Following flushing of the uterine horns the quality of embryos and the fertilization rate were determined. Significant differences between treatments were determined using a two-sided t-test, and frequency distributions were compared using Chi-square tests. The mean number (+/-SEM) of ovulations for heifers in the Day-10 group was 12.9+/-1.0, and 8.5+/-0.9 embryos were recovered. Both the number of ovulations (6.7+/-0.8) and embryos recovered (4.1+/-0.6) were lower (P=0.0001) in heifers in the Day-2 group. Following grading based on a morphological basis, a higher number (P=0.002) of embryos was categorized as Grades 1 and 2 (4.1+/-0.6) and Grade 3 (2.1+/-0.4) in Day-10 heifers than in the Day-2 group (Grade 1 and 2, 1.9+/-0.3; Grade 3, 0.7+/-0.2). The number of Grade 4 and 5 embryos (Day 10, 1.6+/-0.2; Day 2, 1.4+/-0.2) and the number of unfertilized ova (Day 10, 0.7+/-0.4; Day 2, 0.2+/-0.1) did not differ between treatments. Progesterone concentrations were lower (P=0.0001) in Day-2 heifers at FSH treatment prior to prostaglandin, and the decline was more rapid following prostaglandin injection at Day 5 (P=0.02). Results of this study indicate that the number of ovulations and embryos recovered was lower in heifers when FSH treatment was initiated on Day 2 compared with Day 10 of the estrous cycle.     

Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification

The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0.05) were obtained by vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.     

Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen

Precise data on fertility results following peri- and postovulatory insemination in spontaneously ovulating gilts is lacking. Using transcutaneous sonography every 4 h during estrus as a tool for diagnosis of ovulation, the effects of different time intervals of insemination relative to ovulation were investigated with liquid semen (Experiment 1, n=76 gilts) and frozen semen (Experiment 2, n=80 gilts). In Experiment 3 (n=24 gilts) the number of Day-28 embryos related to the various intervals between insemination and ovulation was determined after the use of liquid semen. Using liquid semen the fertilization rates based on Day-2 to Day-5 embryos and the number of accessory spermatozoa decreased significantly in gilts inseminated with 2 x 10(9) spermatozoa per dosage in intervals of more than 12 h before or more than 4 h after ovulation. In the time interval 4 to 0 h before ovulation, comparable fertilization rates were obtained using frozen semen (88.1%) and liquid semen (92.5%). Fertilization rates and numbers of accessory spermatozoa decreased significantly when gilts were inseminated with frozen semen more than 4 h before or 0 to 4 h after the detection of ovulation. The percentage of Day-28 embryos was significantly higher following preovulatory insemination compared to inseminations 0 to 4 h and 4 to 8 h after ovulation. It is concluded that the optimal time of insemination using liquid semen is 12 to 0 h before ovulation, and 4 to 0 h before ovulation using frozen semen. The results stress the importance of further research on sperm transport and ovulation stimulating mechanisms, as well as studies on the time of ovulation relative to estrus-weaning intervals and estrus duration.     

The extent and timing of prenatal loss in gilts

The potential litter size of gilts that is based on the ovulation rate is much higher than the actual litter size, which depends on the fertilization rate and subsequent prenatal mortality. Prenatal mortality is divided into embryonic mortality (before Day 30) and fetal mortality (after Day 30). Prenatal loss includes both fertilization failure and prenatal mortality. Crossbred gilts (n = 149) were bred at the first observed estrus after being exposed to the boar at 200 days of age. Time of the first insemination after estrus detection was determined by measurement of vaginal conductivity using a Walsmeta meter. A second insemination was administered either 8 or 16 hours later. Artificial insemination with fresh semen (0 to 3 days old) was used throughout the experiment. Gilts were slaughtered on Day 3 (n = 26), Day 10 (n = 42), Day 30 of gestation (n = 45) or they were allowed to farrow (n = 36). Gilts slaughtered on Day 3 were used to estimate the fertilization rate. Gilts slaughtered on Day 10 and Day 30 were used to calculate embryonic mortality, while fetal mortality was calculated from the gilts that farrowed. The mean (+/-SEM) number of corpora lutea (CL) was 13.15+/-0.46, 13.36+/-0.37 and 12.97+/-0.39 for gilts slaughtered at Days 3, 10 and 30, respectively (P>0.05), and the mean (+/-SEM) number of normal embryos recovered was 11.12+/-0.69, 9.46+/-0.55 and 9.33+/-0.58, respectively. Litter size at parturition was 9.10+/-0.54. There was a significant difference between the number of normal embryos on Day 3 and Day 30 (P=0.05) and also between the number of normal embryos at Day 3 and the number of piglets at term. Ninety percent of the ova were recovered at Day 3. The fertilization rate was calculated either 1) assuming that unrecovered ova had a similar fertilization rate as the recovered ova (FRER=94.5+/-2.0%) or 2) assuming that unrecovered ova were unfertilized (FROR=84.5+/-2.5%). It was concluded that FRER was a more accurate estimation of the fertilization rate. Based on this fertilization rate, embryonic mortality between Day 3 and Day 10 was 20.8+/-8.3%, with an additional 12.5+/-7.1% loss between Day 10 and Day 30, when all gilts were included (P = 0.308). Thus the total prenatal loss, including fertilization failure, up to Day 10 was 26.3% and to Day 30 it was 38.8%. Fetal mortality was 2.2%, giving a total prenatal mortality (excluding fertilization failure) of 35.5% and a prenatal loss of 41%. Most of the prenatal loss was due to embryonic mortality. In those gilts that remained pregnant most of the embryonic loss occurred before Day 10 (19.0+/-6.3%; P=0.003). There was no further loss between Day 10 and 30 of pregnancy. There was a significant difference between the loss from Day 3 to Day 10 compared with the loss from Day 10 to Day 30 (P=0.05); therefore, most of the embryonic loss in pregnant gilts occurred before Day 10. Since fetal mortality was 3.2+/-6.3%, most of the prenatal loss was due to embryonic mortality.     

Influence of sexual maturity of donor on in vivo survival of transferred porcine embryos.

A study was conducted to determine whether embryos recovered from first-estrous (pubertal) and second-estrous gilts differed in survival when transferred to first- or third-estrous recipients. Embryos were recovered surgically from first- and second-estrous donors 48-72 h postmating and 6-10 normal embryos/zygotes (1-4 cells) were transferred to oviducts (3-5 embryos/ampulla) of nonmated synchronous first- (n = 40) or third- (n = 15) estrous recipients. Blood samples were collected from the jugular vein of recipient gilts on Days 3, 12, and 30 of gestation and the sera were analyzed for progesterone and free (unconjugated) estrogens by use of radioimmunoassays. Recipient gilts were subsequently slaughtered between Days 30 and 40 to assess embryonic losses. Mean number of ovulations was lower among first-estrous vs. third-estrous recipients (8.9 +/- 0.7 vs. 11.4 +/- 0.7; p < 0.05). Percentage of recipients that maintained pregnancy was similar between first- and third-estrous gilts (67.5 vs. 60.0%) and recovery of total conceptuses (normal and degenerating) resulting from transfer of one-cell- and cleavage-stage embryos did not differ among first- vs. third-estrous gilts (76.1 vs. 78.2%). Similarly, percentage of viable fetuses in first-estrous gilts that were pregnant from transfer of one-cell- and cleavage-stage embryos was not different from that of third-estrous gilts (69.3 vs. 75.6%). Percentages of total conceptuses and viable fetuses in first- and third-estrous gilts that were recipients of cleavage-stage embryos only also did not differ (p > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Embryo survival and conceptus growth after reciprocal embryo transfer between Chinese Meishan and Landrace x Large White gilts

Embryos were transferred between Meishan and Landrace x Large White (control) gilts on Day 4 or 5 to establish approximately equal numbers of all four possible combinations of donor breed and recipient breed. The breed of the donor gilt significantly (P less than 0.01) affected embryo survival with 44.5% of transferred Meishan embryos and 69.6% of transferred control embryos surviving to Day 30 +/- 1. There was no influence of the breed of the recipient gilt on the proportion of embryos which survived. These differences in embryo survival between the two breeds could not be explained by differences in (1) the number of embryos transferred, (2) the stage of development of the embryos transferred, (3) the interval between ovulation and transfer or (4) the degree of asynchrony between donor and recipient gilt. On Day 30 +/- 1 embryos from control donors developed into longer fetuses (P less than 0.01) with larger allantoic sacs (P less than 0.05) than did embryos from Meishan donors. Fetuses in control recipients were longer (P less than 0.01), heavier (P less than 0.001) and had larger allantoic sacs (P less than 0.05) than fetuses occupying Meishan uteri. The interaction between breed of donor gilt and breed of recipient gilt did not significantly affect conceptus growth. These results suggest that Meishan pig embryos may be less tolerant to routine embryo transfer procedures than those of control gilts, that the genotype of the dam does not affect the proportion of embryos surviving to Day 30 +/- 1, and that both fetal and maternal factors affect conceptus growth.     

Intrauterine migration of the porcine embryo: influence of estradiol-17 beta and histamine

The role of estradiol-17 beta (E2) in migration of the porcine embryo was examined (Experiment 1) by observing the distribution of Silastic (polydimethyl siloxane, Medical Adhesive Silicone Type A, Dow Corning) beads impregnated with cholesterol or E2 (n=5 gilts per treatment) after 5 days in utero (Day 12 of the estrous cycle, Day 0=1st day of estrus). Beads impregnated with E2 migrated farther (P less than 0.05) than those impregnated with cholesterol. Twenty additional gilts and sows were used to determine if histamine was involved with intrauterine migration (Experiment 2). On Day 6 of gestation the tip of each uterine horn was exposed and the subserosa of each of 5 gilts was injected with either vehicle, 8 mg of cromolyn sodium (an inhibitor of histamine release) or 8 mg of cromolyn sodium plus 1 mg of histamine. Four days later (Day 10), the excised uterus was examined for migration of embryos. An additional group of 5 gilts received 8 mg of cromolyn sodium on Days 6 and 10 and were examined on Day 12. Results from the second experiment demonstrated that cromolyn sodium treatment alone restricted (P less than 0.05) Day 10 embryos to the tip of the uterine horn but by Day 12 embryos had overcome this restriction. Injection of histamine overcame the inhibitory effects of cromolyn sodium and restored migration of Day 10 embryos. These experiments suggest that both E2 and histamine are involved in intrauterine migration of the porcine embryo. The extent to which these hormones might be interrelated during migration is not fully understood at this time.     

Laparoscopic insemination technique with low numbers of spermatozoa in superovulated prepuberal gilts for biotechnological application

New biotechnologies, such as sperm-mediated gene transfer (SMGT), spermatozoa freezing and spermatozoa sorting have improved the possibilities to produce animals with desirable features. The main problem associated with these technologies is the scarce availability of spermatozoa for insemination. The objective of this study was to develop a laparoscopic insemination (LI) technique in gilt that allows the use of low semen doses resulting in high fertilization rates (FR) and minimal distress to the animal; the efficiency of this technique was compared to conventional artificial insemination (AI). Ten gilts were inseminated 36 h post hCG treatment near both utero-tubal junctions (UTJ) with 1.5 x 10(9)spermatozoa/5 mL per horn and 10 gilts (C) underwent conventional AI. Embryos were collected either at two to four cell stage (LI, n = 5; C, n = 5) for determination of fertilization rate or at day 6 for evaluation of developmental competence (LI, n = 5; C, n = 5). LI gilts showed a slightly higher FR than control animals. In a second trial, 24 gilts underwent LI with varying doses (1.5 x 10(8), 1.5 x 10(7), 1 x 10(7), 5 x 10(6) or 1 x 10(6)) of semen. Two to four stage embryos were collected and FR was evaluated in each tube. FR obtained with the lowest dose was significantly different from that with other dosages (P < 0.05). Embryos were cultured in vitro to blastocyst stages (percentage of blastocysts: 79.2 +/- 3.6%). In a third trial, five gilts were inseminated with semen processed by SMGT technique; both FR (86.1 +/- 9.9%) and transgene protein expression were satisfactory. In conclusion, this study shows that LI can be a useful tool for reducing doses of insemination, without affecting the efficiency of fertilization; this technique could have a wide range of biotechnological applications.     

Embryo recovery and pregnancy rates after the delay of ovulation and fixed time insemination in superstimulated beef cows

The aim of this study was to evaluate the effect of delaying ovulation subsequent to superstimulation of follicular growth in beef cows (Bos indicus) on embryo recovery rates and the capacity of embryos to establish pregnancies. Ovulation was delayed by three treatments using either progesterone (CIDR-B) or a GnRH agonist (deslorelin). Multiparous Nelore cows (n = 24) received three of four superstimulation treatments in an incomplete block design (n = 18 per group). Cows in Groups CTRL, P48 and P60 were treated with a CIDR-B device plus estradiol benzoate (EB, 4 mg, i.m.) on Day-5, while cows in Group D60 were implanted with deslorelin on Day-7. Cows were superstimulated with FSH (Folltropin-V, 200 mg), from Day 0 to 3, using twice daily injections in decreasing amounts. All cows were treated with a luteolytic dose of prostaglandin on Day 2 (08:00 h). CIDR-B devices were removed as follows: Group CTRL, Day 2 (20:00 h); Group P48, Day 4 (08:00 h); Group P60, Day 4 (20:00 h). Cows in Group CTRL were inseminated at 10, 20 and 30 h after first detected estrus. Ovulation was induced for cows in Group P48 (Day 4, 08:00 h) and Groups P60 and D60 (Day 4, 20:00 h) by injection of LH (Lutropin, 25 mg, i.m.), and these cows were inseminated 10 and 20 h after treatment with LH. Embryos were recovered on Days 11 or 12, graded and transferred to synchronized recipients. Pregnancies were determined by ultrasonography around Day 100. Data were analyzed by mixed procedure, Kruskal-Wallis and Chi-square tests. The number of ova/embryos, transferable embryos (mean +/- SEM) and pregnancy rates (%) were as follows, respectively: Group CTRL (10.8+/-1.8, 6.1+/-1.3, 51.5), P48 (12.6+/-1.9, 7.1+/-1.0, 52.3), P60 (10.5+/-1.6, 5.7+/-1.3, 40.0) and D60 (10.3+/-1.7, 5.0+/-1.2, 50.0). There were no significant differences among the groups (P > 0.05). It was concluded that fixed time AI in association with induced ovulation did not influence embryo recovery. Furthermore, pregnancy rates in embryos recovered from cows with delayed ovulation were similar to those in embryos obtained from cows treated with a conventional superstimulation protocol.     

Effects of asynchrony on rabbit blastocyst development

The development of synchronously and asynchronously transferred rabbit morulae (recovered at Day 3 p.c.) and blastocysts (recovered at Day 4 p.c.) was investigated before the anticipated time of implantation. The results obtained with various techniques (evaluation of gross morphology, measurement of diameter, thymidine incorporation, light and electron microscopy) led basically to the same conclusions. Embryos being asynchronously transferred to the uterus of recipient rabbits survived, at least in terms of certain cellular functions like cell proliferation, for more than 2 days. Development, however, was clearly retarded and ultrastructural examination revealed substantial cell damage. Some blastocysts showed, even after 3 days, normal growth and cell proliferation indicating considerable differences between individuals in the ability to compensate for suboptimal developmental conditions before implantation. In general, this ability was greater in the transferred Day-3 morulae than in the Day-4 blastocysts. Embryonic growth and the ability to dissolve the zona pellucida, to synthesize crystalloid bodies and to differentiate extraembryonic endoderm indicated the maintenance of some developmental functions under asynchronous conditions. Blastocyst development was influenced by the progestational stage of the recipient. At 1 day after transfer into asynchronous older uteri, blastocyst diameters were larger and cell proliferation was increased compared with all other groups, suggesting an attempt of the blastocyst to adjust to the more advanced maternal milieu. Development in asynchronous younger uteri was delayed. No comparable differences in development were found in cultured embryos for which the media had been supplemented with flushings from the same progestational uterine stages as used for transfer. Thymidine incorporation in cultured embryos did not differ between the various supplements (P greater than 0.05) and was generally lower than in chronologically aged asynchronously transferred embryos (P less than 0.05 for Day-3 and P less than 0.001 or P greater than 0.05 for Day-4 embryos).     

The effects of administration of gonadotrophins and estrogens on prenatal survival in gilts

In gilts ovulation occurs over a 4 to 8-hour period, with 70% of the ova being shed over a relatively short span of time. These oocytes supposedly give rise to more developed embryos at Days 10 to 12 which advance the uterine environment and reduce survival rates of less developed embryos because of an asynchronous environment. The aim of this experiment was to reduce embryo mortality by influencing the duration and pattern of ovulation. Crossbred gilts (n = 98) were bred at their first observed estrus after being exposed to boars at 200 days of age. Estrus detection was carried out daily at 0000, 0800 and 1600 hours. All gilts were artifically inseminated with fresh semen, with a minimum of 2.7 billion spermatozoa, at both 16 and 32 hours after detection of estrus. Gilts were randomly assigned to one of the following treatments at detection of estrus: 1) 500 IU (2ml) chorionic gonadotrophin (hCG) injected intravenously at the onset of estrus (n = 22); 2) 16 mug (4 ml) gonadotrophin releasing hormone (GnRH) injected intravenously at the onset of estrus (n = 25); 3) 11.5 mug estrogen added to the semen at the time of AI (n = 25); 4) control, untreated gilts (n = 26). All gilts were slaughtered at Day 30 of gestation (Day 0 = day of detected estrus). The mean (+/-SEM) number of ovulations in pregnant gilts per treatment was 13.0 +/- 0.52, 12.6+/-0.51, 13.6+/-0.54 and 13.3+/-0.52, while the mean (+/-SEM) number of normal embryos per treatment was 10.3+/-0.67, 10.5+/-0.66, 10.3 +/- 0.69 and 10.5 +/- 0.67 for hCG, GnRH, estrogen and control groups, respectively, for an embryonic survival rate of 80 +/- 4.2%, 83 +/- 4.1%, 74 +/- 4.3% and 79+/-4.2% in pregnant gilts. If nonpregnant gilts are included, the embryonic survival rate for treatments 1 to 4 was 76+/-7.0%, 73+/-6.5%, 60+/-6.5%, and 64+/-6.4%, respectively. There was no significant difference between treatments for any of these variables. There was no evidence that administration of hCG, or GnRH at the onset of estrus, or the addition of estrogen to semen improved embryonic survival in gilts by Day 30 in this experiment.     

Endoscopic embryo collection and embryo transfer into the oviduct and the uterus of pigs

We describe the first complete embryo transfer program, including flushing of embryos from the oviducts via the uterine horns, transfer of embryos into the Fallopian tubes or the uterine horns and recording of the number of piglets born live. The described procedure is minimally invasive and allows the use of pigs simultaneously for embryo collection and production of normal pregnancies. A 30 degrees forward oblique endoscope provided optimal visualization of the reproductive organs and free access to the organs for embryo flushing and transfer. In contrast to surgical and nonsurgical methods, endoscopy allows to pre-examine the genital tract for reproductive abnormalities and successful ovulation. A total of 95 prepuberal gilts or cyclic sows were used in this trial. Embryos or oocytes were collected from hormonally treated pigs via endoscopy(n = 17) on Day 3 and via laparotomy or post mortem after slaughter (control group, n = 38) on Day 3 and 6 after insemination. One (unilateral collection, n = 7) or both oviducts (bilateral collection, n = 10) were flushed endoscopically. We recovered 114 (average 16/pig) and 279 (average 28/pig) oocytes or embryos with fertilization rates of 89% and 72%, respectively. In the control group 834 oocytes or embryos were collected at Day 3 and 6 after insemination (fertilization rate 64%, total 534 embryos, 33 at 2-, 367 at 4-, 2 at 8-cell stage, 24 morulae and 108 blastocysts). Of 836 embryos recovered by endoscopy, surgery or slaughter 528 Day 3 embryos at 2- to 4-cell stage were transferred into (one) oviducts (n = 27 pigs, about 20/pig) resulting in 9 pregnant pigs diagnosed at Day 28 by sonography. Of the 9, 8 carried a total of 49 piglets to term. A total of 195 Day 6 embryos were transferred into uterine horns (n = 12 pigs, about 16/pig), resulting in 5 pregnant pigs carrying a total of 38 offspring to term. The use of endoscopy in assisted reproduction of pigs has the advantages of allowing easy access to the ovary, oviduct and uterus, clear view of the organ manipulation without exposure and exteriorization of viscera during surgery.     

Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares

In this study we examined the ability of equine oviductal epithelial cells (OEC) to support the development of four- to eight-cell equine embryos in vitro and investigated the ability of co-cultured embryos to continue normal development after transfer to synchronous recipient mares. Equine embryos obtained at Day 2 after ovulation were cultured with or without OEC for 5 days. Those OEC co-cultured embryos that reached the blastocyst stage and embryos recovered from the uterus at Day 7 were surgically transferred to synchronous recipient mares. Co-culture with OEC improved (P < 0.01) development of four- to eight-cell embryos to blastocysts compared to medium alone (11/15 vs 0/6) during 5 days in vitro. Embryos co-cultured with OEC were smaller (P < 0.05) and more delayed in development than Day-7 uterine blastocysts. There was no difference in the Day-30 survival rate of co-cultured blastocysts (3/8) or Day-7 uterine blastocysts (5/8) after transfer to recipient mares. These results indicate that co-culture with OEC can support development of four- to eight-cell equine embryos in vitro and that co-cultured embryos can continue normal development after transfer to recipient mares.     

Effects of conditioned media on porcine embryos at different stages of development

The objective of our study was to determine the effect of conditioning media with homologous porcine uterine cells on the developmental rate of porcine embryos. Cell monolayers were prepared by selective dissection and digestion of sections from the uterus of prepuberal gilts that were primed with PMSG and hCG. Conditioned media were used with 2 type of embryos: 4-cell stage (Experiment 1) or blastocyst stage (Experiment 2). In Experiment 1, embryos were collected surgically by flushing the oviducts, 36 to 48 h following the first of 2 inseminations. Embryos were cultured in Whitten's medium containing 1.5% BSA as a protein source until they attained the 4-cell stage. Embryos at the 4-cell stage were cultured randomly in either Whitten's medium with 1.5% BSA or Whitten's medium with 1.5% BSA that was previously conditioned for 24 h with an endometrial epithelial cell monolayer. Embryos were cultured in 50-microl drops covered with oil in a 38.5 degrees C, 5% CO(2) in air incubator. There was no advantage to using the conditioned media with the 4-cell stage embryos. The embryos were less developed than those cultured in nonconditioned Whitten's medium (P <0.001). In Experiment 2, embryos were cultured at the blastocyst stage. They were recovered the same way as in Experiment 1 and then cultured in Whitten's medium containing 1.5% BSA until they reached the blastocyst stage. At the blastocyst stage (Day 6), embryos were randomly assigned to 1 of the 6 following treatments: Whitten's with 1.5% BSA or Whitten's plus 1.5% BSA that was previously conditioned with endometrial epithelial cell monolayer, TCM-199 containing 0.4% BSA or TCM-199 plus 0.4% BSA that was previously conditioned with endometrial epithelial cell monolayer, finally, TCM-199 containing 10% serum or TCM-199 plus 10% serum that was previously conditioned with endometrial epithelial cell monolayer. Results show that initiation of hatching was significantly enhanced by conditioning the Whitten's media.