Culture of pig embryos collected in situ or after slaughter

Two-cell embryos collected from synchronized prepuberal gilts either by surgical means or after slaughter were incubated in Dulbecco's medium supplemented with 20% inactivated bovine blood serum. Embryo recovery rate in situ was 89%, when flushing oviducts from slaughtered animals it was 80%.When culturing 61 two-cell embryos collected in situ, 80.3% continued to develop. Out of 63 two-cell embryos obtained after slaughter, only 44.4% continued to develop. Hardly any of the embryos developed beyond the four-cell stage.     

Bovine oocyte maturation, fertilization and further development in vitro and after transfer into recipients

Bovine oocytes were aspirated from ovaries within 1.6 to 2 hours after slaughter. They were then matured in TCM-199 medium drops under oil in CO(2)/air incubator at 39 degrees C. Spermatozoa were capacitated in SP-TALP medium with heparin. The percentage of embryos that developed in vitro to the 4- and 6- cell stages 48 hours post insemination and then reached the morula or blastocyst stage was 64.3% and 59.2%, respectively, while only 3.6% of the embryos that reached the 2-cell stage became morula or blastocysts. An average of 6.3+/-3.2 total in vitro fertilized embryos per cow were obtained (range 2 to 11). Maturation of bovine oocytes in vitro for 18 or 24 hours did not influence the percentage of cleaved embryos (71.0 and 75.9%, respectively) or that developed to the blastocyst stage (25.6 and 24.2%, respectively). The use of reindeer blood serum for in vitro culture of immature bovine oocytes and of dividing of embryos gave the following results: 57.4% of the oocytes cleaved after fertilization and 16.2% developed further to the blastocyst stage. In contrast in the control group, where cow serum was used, the values were 73.4% and 24.8%, respectively. Rabbit oviduct epithelium cell monolayers were able to support the development of 16.3% of the cleaved bovine embryos to the blastocyst stage as compared with 24.0% of the embryos on cow oviduct epithelium cell monolayers. After nonsurgical transplantation, 12 calves were produced from 91 in vitro fertilized embryos.     

Comparison of embryonic developmental competence of mouse oocytes grown with and without serum.

The first objective of this study was to determine whether oocyte growth in serum-free medium affects the solubility of the zona pellucida to alpha-chymotrypsin digestion, which is an index of zona pellucida "hardening" and reflects the potential penetrability of the zona pellucida by sperm. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in medium containing 5% fetal bovine serum (FBS) or in serum-free medium. The zonae pellucidae of oocytes grown in serum-free medium were four times as hard as freshly isolated germinal vesicle (GV)-stage oocytes grown in vivo or oocytes grown in vitro in FBS-containing medium. The hardening of the zonae pellucidae of oocytes grown in serum-free medium was prevented by addition of fetuin. The second objective was to compare the competence to undergo embryogenesis of oocytes that grew in serum-free vs. FBS-containing medium. Approximately 70% of the oocytes underwent maturation regardless of whether the medium was serum-free or contained FBS. Of the mature ova grown in medium containing FBS, 53% cleaved to the two-cell stage after insemination compared with only 6% of the ova grown in serum-free medium. Addition of fetuin to the serum-free medium used for oocyte growth increased the frequency of cleavage to the two-cell stage. Of the embryos derived from oocytes that grew in FBS-containing medium, 70% completed the two-cell stage to blastocyst transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recovery rate and quality of embryos collected from suckled cows and beef heifers after superovulation with PMSG

Recovery rate and embryo quality were investigated in beef heifers and suckled cows following superovulation induced by 2000 IU pregnant mare serum (PMSG) combined with different methods of estrus cycle synchronization (Norgestomet, Prid, Dinolytic, Norgestomet combined with Dinolytic). Genital tracts were flushed upon slaughter with Dulbecco's medium 6.5 to 7.5 days after insemination. Of the heifers, 42 out of 43 responded to treatment. The mean embryo recovery rate, based on the number of corpora lutea, was only 14.8%. Of the 83 embryos recovered, 54.2% had developed to the expected stage and only 40% appeared normal. Of the adult cows, 55 out 58 responded with an embryo recovery rate of 39.5%. Of the 149 embryos recovered, 48.9% had developed to the expected stage and 67.1% of these appeared normal. In both heifers and adult cows, the different methods of estrus synchronization produced no significant differences in recovery rate or embryo quality.     

Effect of six-and ten-day-old chick embryo amniotic fluid on development of two-cell mouse embryos.

The purpose of this study was to investigate the effect of six-and ten-day-old chick embryo amniotic fluid (CEAF) on the development of two-cell mouse embryos. Six- and ten-day-old CEAF (6-AF, 10-AF) were aspirated separately from the amniotic cavity and two experiments were performed. In the first experiment, two-cell mouse embryos were cultured in different supplements of heat-inactivated 6-AF, pure heat-inactivated 6-AF and pure active 6-AF. The second experiment was also carried out in the same manner using the 10-AF. The rate of the development of embryos in all groups were daily determined and statistically compared with that of control (Ham's F-10 supplemented with 5 mg/ml human serum albumin). During the cultivation period, more embryos reached to the blastocyst stage in all groups of CEAF compared to the control. The result hatched blastocysts embryos also increased from days 3 till 5 of cultivation in all groups of CEAF. The developmental potential of embryos appears to be almost identical in six- and ten-day-old CEAF. In conclusion, CEAF as a supplement or a natural medium could support the development of two-cell mouse embryos.     

Effect of human leukemia inhibitory factor on in vitro development of parthenogenetic bovine morulae

The present study was conducted to investigate the effect of human leukemia inhibitory factor (hLIF) addition to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HS) on the development of in vitro matured and parthenogenetically activated bovine oocytes. The oocytes matured for 30 h were exposured to ethanol (7%, 7 min) and cytochalasin B (5 mug/ml, 5 to 6 h). The treated oocytes were cultured for 5 d in SOFM supplemented with HS, and Day-5 morulae were cultured for 2 d in SOFM supplemented with HS and with or without hLIF (5000 U/ml) to investigate the subsequent in vitro development to the blastocyst stage. Of the 1531 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to 8-cell stage and 174 (13.8%) developed to the morula stage. The addition of hLIF at the morula stage resulted in a significantly (P<0.01) higher rate of development to the blastocyst stage in the medium with hLIF (55.9%) than without hLIF (28.9%). The mean cell number per blastocyst developed in the medium with hLIF was also significantly (P<0.01) higher than that developed in the medium without hLIF. To evaluate the viability, 6 parthenogenetically developed blastocysts were transferred to 3 recipient heifers (2 embryos per heifer), while in 2 other recipient heifers estrus was prolonged after transfer. The plasma progesterone levels of the 2 recipient heifers at the 28th day after transfer were 8.1 ng/ml and 9.0 ng/ml, but pregnancy was not observed by ultrasonic scanning. The present results indicate that the addition of hLIF to in vitro-produced, Day-5 parthenogenetic bovine morulae significantly improves the subsequent development to the blastocysts stage; however, the present method still does not promote for development of parthenogenetic fetuses in cattle.     

Porcine oviductal cells support in vitro bovine embryo development

This study was designed to investigate the developmental competency of in vitro-matured and in vitro-fertilized bovine embryos co-cultured with a) medium alone, b) bovine oviductal cells (BOC), c) bovine conditioned medium (BCM), d) porcine oviductal cells (POC), and porcine conditioned medium (PCM). Follicular oocytes collected from cattle at local slaughterhouses were matured and fertilized in vitro. Epithelial cells were scraped from the luminal surface tissue of either bovine or porcine oviducts collected after ovulation, cultured in TALP + 10% heat-treated fetal calf serum, and the conditioned media were collected following a 3- to 5-d incubation period. After 18 to 22 h of sperm-ova co-incubation, the fertilized and/or cleaved ova were randomly assigned to 1 of 5 co-culture groups. The results revealed that the efficiency of medium alone in supporting embryo development from the 16- to 32-cell stage up to the blastocyst stage was significantly (P<0.01) lower than of embryos co-cultured with either bovine or porcine epithelial cells, or with conditioned media from such cells. Epithelial cell co-culture, regardless of cell source, was more effective (P<0.01) than culture with conditioned medium. Co-culture in medium containing or conditioned by porcine cells was more effective in supporting bovine embryo development than co-culture with bovine-derived cells or conditioned medium. These data support the concept that oviductal cells produce a soluble component which enhances embryo development to the blastocyst stage in vitro and that the effect is not species-specific.     

The effect of bovine pestivirus infection on the superovulatory response of Friesian heifers

The pathogenesis of reproductive loss associated with bovine pestivirus infection during the preovulatory period was investigated using superovulated heifers. Twenty-five Friesian heifers were selected and randomly assigned to either a control group (n = 12) which did not become infected or to a treatment group (n = 13) which became infected following intranasal instillation of 2 ml of serum inoculum containing 5.5 log(10) TCID(50)/ml non-cytopathic virus, 9 d prior to artificial insemination (AI). Transrectal ultrasonography was used to monitor follicular development and ovulation during the superovulatory period. Animals were superovulated using a standard protocol of twice-daily injections of FSH-P and then were inseminated twice commencing 12 h after the onset of estrus. The intensity of expression of estrus was higher in the control heifers than in the pestivirus-infected heifers. Of 13 pestivirus-infected heifers, only 3 heifers displayed standing estrus compared with that in the control group, in which 10 of 12 heifers exhibited standing estrus. The mean number of ova/embryos recovered from the control group heifers was 5.75 +/-2.31, of which 4.00 +/- 0.72 were evaluated as transferable quality embryos. In comparison, heifers in the pestivirus-infected group yielded only a mean of 0.60 +/-0.34 ova/embryos, of which 0.23 +/- 0.22 were transferable quality embryos. Based on ultrasonographic examination, 24 h after the first AI 82% of the presumptive ovulatory follicles had ovulated in the control group compared with an ovulation rate of only 17% in the treated group. The results of this experiment demonstrated that bovine pestivirus infection during the preovulatory period could adversely affect ovulation, thus leading to a significant reduction in the number of palpable corpora lutea and in the number and quality of embryos recovered.     

Culture of one- and two-cell bovine embryos to the blastocyst stage in the ovine oviduct

The ovine oviduct was evaluated as a culture system for early bovine embryos. One- to two-cell embryos were collected from superovulated heifers killed 36 or 48 h after the onset of estrus, embedded in agar cylinders, and transferred to oviducts ligated at the uterotubal junction. After 5 d (6.5 to 7.0 d after donor estrus), embryos were recovered and evaluated for development to the late morula or blastocyst stage. In Experiment 1, 86 embryos were cultured in 10 ewes in which the onset of estrus was synchronized with that of the donors. Fifty-eight embryos (68%) were recovered; of these, 31 (53%) had continued normal development. In Experiment 2, development in ovariectomized versus intact cyclic ewes was compared. Recovery from ovariectomized ewes (26/39, 67%) did not differ from intact cyclic ewes (26/35, 74%) and the proportion developing normally also did not differ (ovariectomized: 7/26, 27%; intact cyclic: 11/26, 42%). In Experiment 3, embryo development was compared in anestrous versus ovariectomized ewes. Recovery rate (anestrous: 22/43, 51%; ovariectomized: 20/51, 39%) and the proportion developing normally (anestrous: 8/22, 37%; ovariectomized: 9/20, 45%) did not differ between treatments. Developmental competence of oviduct-cultured embryos was tested by transfer to 16 synchronous heifers, of which eight (50%) became pregnant; five delivered calves. Results indicate that the ovine oviduct provides an adequate site for the culture of early bovine embryos.     

The efficiency of production, centrifugation, microinjection and transfer of one- and two-cell bovine ova in a gene transfer program

Forty crossbred beef heifers were superovulated with 2000 IU pregnant mare serum gonadotropin (PMSG) and mated twice by natural service during estrus. Ovulations were counted and ova were recovered during mid-ventral laparotomy between 44 and 54 h after the onset of estrus. The overall donor ovulation rate (M+/-SEM) was 15.2+/-1.3. There was a positive association between ovulation rate and the number of ova recovered (P<0.001), and between ovulation rate and the incidence of ova advanced beyond the two-cell stage of development (P<0.05). When grouped on the basis of superovulation response, the numbers (M+/-SEM) of recovered one-cell, two-cell and more advanced ova were 3.7+/-0.7, 1.0+/-0.3 and 0.5+/-0.3, respectively, for donors with up to 15 ovulations. The corresponding numbers for donors with more than 15 ovulations were 7.2+/-1.8, 6.0+/-1.3 and 2.8+/-1.2, respectively. Following centrifugation, pronuclei were visible in 68% of one-cell ova, and nuclei were visible in 80% of two-cell ova. Approximately 20% of ova were destroyed during DNA microinjection. A total of 66 centrifuged and DNA-injected ova were transferred to the oviducts of 26 crossbred beef heifers, each receiving two, three or four ova. Echography at Day 55 confirmed that 14 (54%) heifers were pregnant with 26 (39%) fetuses. Eleven heifers were held to calve and produced 21 calves.     

A mouse embryo culture system for quality control testing of human in vitro fertilization and embryo transfer media and fetal cord sera

Swiss white mice were superovulated, mated, and sacrificed to recover two-cell embryos that were cultured in Ham's F-10 supplemented with 15% fetal serum. In 16 experiments, media enriched with fetal bovine serum (FBS) supported blastocyst development from 80% ± 19% (mean ± S.D.) of two-cell embryos. Culture media + FBS was the positive control when 74 batches of heat-inactivated human fetal cord serum (hFCS) were tested. Statistical analyses indicated two distinct populations: 49 hFCS promoted blastocyst formation and 25 hFCS grew fewer blastocysts. In five studies, 35/47 two-cell embryos recovered from mice oviducts in media + FBS and immediately incubated formed blastocysts (75% ± 10%). In six comparison studies where the recovered embryos stood at room temperature for 30 minutes before incubation, only 18/57 (29% ± 21%) became blastocysts. When the colony was housed for 1 week in rooms with Shell No Pest Strips as treatment for mites, only 11/125 two-cell embryos became blastocysts (9%). In contrast, animals housed in quarters decontaminated with chlorine bleach had reduced breeding efficiency and produced fewer two-cell embryos. We conclude that (1) Ham's F-10 + FBS is an excellent positive control to test new batches of hFCS; (2) hFCS that supports blastocyst formation from ≥75% of two-cell embryos is adequate for human use; (3) pesticide treatment of breeding colonies and cooling of murine embryos during harvest both impaired in vitro blastocyst development; and (4) chlorine bleach cleansing of animal quarters reduced the number of successful matings.     

Pregnancies after in-vitro fertilization of cow follicular oocytes, their incubation in rabbit oviduct and their transfer to the cow uterus

Cow embryos fertilized in vitro (1-8-cell) (n = 113) were transferred surgically to the ligated oviduct of pseudopregnant female rabbits (31 +/- 4 h after 75 i.u. hCG). Rabbits were killed 99 +/- 5 h later and 67 embryos were recovered: 45 (67%) had cleaved at least once, 15 had reached the morula stage and 2 blastocysts were obtained. Transfer of cleaved embryos (2-8-cell) led to a high recovery rate (84%) compared to 39% for one-cell embryos. Of the embryos incubated for more than 99 h in the rabbit oviduct, 41% were at the morula stage. Embryos incubated in vivo (n = 21) (8-cell to blastocyst) were transferred to the uterus of 14 synchronized recipient heifers by a surgical (N = 5) or a non-surgical (N = 9) procedure: 6 pregnancies resulted (4 from the non-surgical procedures). In addition, 27 (2-8-cell) cow embryos developed in vitro were transferred to the oviduct of synchronized heifers by a paralumbar surgical approach on a standing animal, but no pregnancies resulted. It is therefore concluded that (1) the rabbit oviduct can be used to obtain cow eggs at an embryonic stage sufficiently advanced to permit transfer to the uterus of a synchronized recipient; and (2) the pregnancy rate after in-vitro fertilization and incubation in the rabbit oviduct are similar to results with fertilization in vivo.     

Triglyceride content of bovine oocytes and early embryos

A microfluorescence technique was used to measure the triglyceride content of a minimum of two bovine oocytes or preimplantation embryos up to the hatched blastocyst stage. Embryos were produced in vitro from abattoir-derived ovaries and grown in medium containing synthetic oviductal fluid, amino acids and BSA (SOFaaBSA medium); 10% fetal calf serum was added to some of the embryos at the four-cell stage. Before maturation, the triglyceride content of oocytes was 59 +/- 1.37 ng and it decreased (P < 0.05) after maturation to 46 +/- 0.85 ng. A decrease in triglyceride content (P < 0.05) was also observed after fertilization with the formation of the two-cell embryo (34 +/- 1.80 ng). In the absence of serum, the triglyceride content remained relatively constant from the two-cell to the hatched blastocyst stage. The triglyceride content of blastocysts produced in vivo was similar (33 +/- 0.70 ng) to that of blastocysts produced in vitro in the absence of serum. In contrast, the triglyceride content of embryos grown with 10% fetal calf serum increased steadily from the 9-16-cell stage to a value in hatched blastocysts (62 +/- 1.14 ng) almost double that in serum-free conditions. These results indicate that triglyceride may act as energy source during bovine oocyte maturation and fertilization and that the presence of serum causes excessive synthesis or accumulation of triglyceride in early embryos.     

Embryos derived from the in vitro fertilization of oocytes of pregnant cows

The aim of our study was to determine if the oocytes of pregnant cattle are capable for undergoing embryonic growth following in vitro fertilization. The ovaries of nine heifers at 4 to 7 months of pregnancy were collected at an abattoir and transferred to the laboratory. A total 191 oocytes (10.6 per ovary) collected by aspiration were matured and fertilized by frozen-thawed semen. Embryos were co-cultured with granulosa cells in modified TCM 199 medium and 20% estrous cow serum. The cleavage rate of embryos was 48%, and 41% of of the cleaved embryos developed to the morula/blastocyst stage 7 days after insemination. Additionally, the ovaries of 10 nonpregnant heifers were also collected, yielding 213 oocytes (10.7 per ovary). The cleavage rate was 51%, and 35% of those which cleaved reached the morula/blastocyst stage. No significant differences were found between the two groups. The average number of transferable-stage embryos obtained from pregnant and nonpregnant animals was 4.1 and 3.7, respectively. Our results indicate that preganancy does not influence the meiotic competence of bovine oocytes, and transferable stage embryos can be obtained by the fertilization of oocytes derived from pregnant animals.     

Embryo transfer in cattle experience of twenty-four completed cases

Thirty-nine potential embryo transfer and 13 ovum collection experiments were initiated within a small experimental herd of mainly nulliparous cattle. Donors for 50 of the experiments were treated with pregnant mares' serum gonadotrophin (PMSG) with or without human chorionic gonadotrophin (HCG) to induce superovulation. Optimal responses (3–20 ovulations) occurred in 56.4 % of treated animals; there was some evidence of reduced responsiveness to repeated treatment. Overall ovum and embryo recovery rate at slaughter or laparotomy was 46.3 % but in some experiments exceeded 80 % on Days 4 and 5 after estrus. Recovery rates were adversely affected by previous surgery and by excessive ovulation rates. 73% of recovered ova were fertilized. Only 20 of the 39 potential transfer experiments were completed, 15 of the 19 incomplete experiments being due to donor failures (lack of superovulation in 11 cases, no fertilization in four). Transfers of three embryos (one case), two embryos (19 cases) or one embryo (four cases) were made surgically to 24 recipients. Of the first 10 recipients, one conceived; of the last 14, ten (71.4%) did so. Best, results were obtained by collecting ova on Day 5 or 6 and using recipients within one day of being synchronous with the donor. Embryos collected at slaughter gave results as good as did those collected surgically. Ten of the pregnant recipients had received more than one embryo but only four gave birth to more than one calf. Some problems associated with the practical application of this technique are discussed.     

Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro

The survival and developmental capacity of cumulus cell-enclosed oocytes frozen (1) at the germinal vesicle (GV) stage, after maturation in vitro with (2) and without (3) FSH, and (4) after gonadotrophin-stimulated ovulation were assessed. Survival, defined as the number of morphologically normal oocytes, after freeze-thaw at the GV stage (69%), was lower than for oocytes frozen after ovulation (84%), and after maturation in vitro with FSH (88%) and without FSH (81%). Treatment with DMSO without freezing had no effect on survival when compared with untreated controls except in immature GV-stage oocytes for which there was a slight reduction. After insemination in vitro, 9% of frozen-thawed GV-stage oocytes cleaved to two equal blastomeres, but none developed to blastocysts. Of oocytes matured in vitro before freezing, 17% cleaved to the 2-cell stage and 18% of these developed to blastocysts. When oocytes were matured in vitro in the presence of FSH, however, the percentage cleaving to the 2-cell stage after freeze-thaw was improved to 55%, and 77% of 2-cell stage embryos developed to blastocysts. When ovulated cumulus cell-enclosed oocytes were frozen, 88% cleaved and 67% of the cleaved embryos developed to blastocysts. When 158 two-cell embryos resulting from oocytes matured in vitro with FSH were transferred to the oviducts of pseudopregnant foster mothers, 41 genetically marked live young were produced (26%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Maturation, fertilization and development of bovine oocytes in vitro using TCM199 and a simple defined medium with co-culture

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39 degrees C under 5% CO(2) in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69 125 ), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (65 69 ), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.     

Development of in vivo derived diploid and tetraploid pig embryos in a modified medium NCSU 37

The aim of this study was to assess development of diploid and tetraploid in vivo derived pig embryos cultured in a modified medium NCSU 37 in an atmosphere with reduced concentration of oxygen. The tetraploid embryos were produced by electrofusion of two-cell embryos that had been cultured in vitro from the one-cell stage before fusion (cultured two-cell embryos) or by fusion of freshly recovered two-cell embryos. Development to blastocyst stage of tetraploid embryos, generated from the cultured two-cell embryos was significantly inferior to the development of control one-cell embryos (29.1 +/- 9.7% versus 66.8 +/- 9.7%; P < 0.05). However, development of tetraploid embryos produced from the freshly recovered two-cell embryos and control two-cell embryos was very similar (89.9 +/- 6.1% versus 81.3 +/- 3.4%). Detection of chromosomes 1 and 10 by in situ hybridization showed that more than 85% of the cultured control embryos were diploid while 15% of the embryos were mosaic. Among the fused embryos 50% were tetraploid, 29% mosaic and 21% diploid. These data indicate that the modified medium NCSU 37 provides optimum environment for pre-implantation development of pig diploid and tetraploid embryos.     

Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells

One-cell bovine embryos fertilized in vivo were cultured in TCM-199 and bovine oviductal epithelial cells, in TCM-199, or in explanted immature mouse oviducts supported by TCM-199 to compare development to the blastocyst stage. The morphological stage of development and cell number were determined following 144 hours of culture. Of the embryos that cleaved at least once, 52.6, 30.4 and 0.0% developed to the morula/blastocyst stage after culture in oviductal epithelial cells, in TCM-199 alone, or in explanted mouse oviducts, respectively. The mean total cell number for embryos cultured in oviductal epithelial cells (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<0.05). The mean cell number of embryos cultured in TCM-199 or in explanted mouse oviducts did not differ. The explanted immature mouse oviduct supported by TCM-199 did not provide an environment adequate for development of one-cell bovine embryos to the blastocyst stage. Development of one-cell bovine embryos was best supported by co-culture with oviductal epithelial cells in TCM-199 medium.     

Invitro culture and maintenance of ovine embryos transported in Dulbecco's enriched phosphate-buffered saline

An initial comparative evaluation was made on the response of ovine morulae and blastocysts cultured in Dulbecco's PBS enriched with either 20% fetal calf serum (FCS). 20% neonatal lamb serum (NLS) or 20% lamb serum (LS). There were no significant differences (P≤0.05) in embryo development in these sera, except that the blastocysts hatched only in PBS plus FCS. Sixty percent of the morulae and 100% of the blastocysts continued to develop within 24 hr of culture. Based on these data, PBS plus FCS was selected as the transport medium. There was a significant decrease (P≤0.05) in the development of embryos transported in PBS plus FCS. Firty-five percent of the 298 morulae and blastocysts transported underwent development within 22 to 27 hr, in contrast to 83% of those maintained under similar culture conditions within the laboratory. Of those embryos that developed further during transport, 54% resulted in a lamb, whereas of embryos that remained visually unchanged, only 9% developed to term. The overall lambing rate of all morulae and blastocysts transported in PBS plus FCS was 0.42.