Proteins associated with the early intrauterine equine conceptus.

A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16-17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F(2 alpha) (PGF(2 alpha)). Uterocalin and beta(2)-microglobulin (beta(2)M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were similar in conceptuses from control and treated mares. A 17 kDa cationic protein identified as a secretory phospholipase A2 (sPLA2) type IIA was detected bound to normal capsules but increased substantially in response to PGF(2 alpha). Two forms of uteroglobin were distinguished by partial amino acid sequences of approximately 6 kDa bands in flush fluids from normal pregnant uteri. After administration of PGF(2 alpha) one immunoreactive form of uteroglobin was preferentially increased. These studies demonstrate that failure of pregnancy in this model is associated with an increase in secretory phospholipase in the capsule and a change in the forms of uteroglobin in the uterine secretions.

Equine embryology: an inventory of unanswered questions.

Carl Hartman's title of 47 years ago is invoked in tribute to his first recovery of a bovine embryo 30 years before that, and his legacy of an emphasis on the value of descriptive and comparative studies in reproductive biology. The horse's qualification as a farm animal has waned since those times but, in a conference understandably dominated by research in ruminants and pigs, there are lessons to be learned from some peculiarities of equine embryonic development. Morphological and physiological features of the conceptus and its interaction with its environment during the first month of pregnancy are described and discussed, with emphasis on conceptus expansion, experimental study of the capsule and its associated proteins, and steroid production and metabolism by the various tissues within the conceptus. It is also suggested that easy access to entire conceptuses at advanced stages of development in horses offers valuable opportunities for comparative investigation of early organogenesis and fetal membrane differentiation and, possibly, how they are affected by embryo manipulation in vitro.

Assessment of immunoregulation by cultured, pre-attachment bovine embryos.

The possibility that the viability of bovine embryos might be predicted by measuring their release of immunoregulatory substances during culture has been investigated. Bovine embryos between days 2 and 19 of gestation were cultured for 24-48 h, the embryo-conditioned medium was harvested and studied for suppression of PHA-stimulated bovine leukocyte cultures. Medium incubated in the absence of any conditioning tissues served as control. Artefactual immunosuppression was detected in incubated control material that could be attributed, in part, to the mixing of different tissue culture media, the type of plastic-ware employed for incubation and supplementation of media with additional L-glutamine. It was observed that day-2 to day-9 bovine embryos, cultured in medium able to support the lymphocyte proliferation assay, did not release immunosuppressive substances. Medium conditioned by day-10 to day-12 embryos produced variable immunosuppression while that conditioned by trophoblastic vesicles derived from day-14 to day-19 embryos was consistently highly suppressive. Since bovine embryo transfer is normally conducted at 6-8 days of gestation, it is unlikely that measuring the immunosuppressive products released from bovine embryos will be of value for predicting their viability.

Osmolality of equine blastocyst fluid from day 11 to day 25 of pregnancy.

Horse conceptuses collected between Day 11 and Day 18 of pregnancy float in isotonic media. To investigate this phenomenon, blastocyst fluids from 30 conceptuses from 13 mares were analysed for osmolality and for concentrations of Na+, Cl-, K+, glucose, urea and creatinine. In conceptuses from Group A, samples from Day 11 to Day 16 yielded the following results (mean +/- s.e.m.): osmolality, 121.4 +/- 1.5 mOsm kg-1; Na+, 11.0 +/- 2.2 mM; Cl-, 29.3 +/- 2.5 mM; K+, 26.2 +/- 2.6 mM; glucose, 0.6 +/- 0.1 mM; urea, 6.0 +/- 0.6 mM; creatinine, 9.6 +/- 1.1 microM. Between Day 16 and Day 25, the osmolalities and Na+ concentrations increased gradually with age but the former never exceeded 255 mOsm kg-1. Fluids from Group B were obtained from eight conceptuses exposed to saline solutions of different osmolalities for various periods of time. An increased perivitelline space of 1-3 mm became evident at the lower pole of the floating conceptus after 45 min of exposure to solutions with osmolalities of > or = 300 mOsm kg-1, suggesting that Na+ and Cl- diffuse freely through the capsule but not through the trophoblast. The significance of the hypo-osmolality of equine blastocyst fluid is discussed but remains unclear.

Developmentally related changes in the uptake and metabolism of glucose, glutamine and pyruvate by cattle embryos produced in vitro.

The metabolism of, and retention of radioactivity from, radiolabelled glucose, glutamine and pyruvate were measured in individual cattle embryos produced in vitro from the 2-cell to hatched blastocyst stage. Uptake was defined as the numeric sum of metabolism and retention of radiolabel. Glucose metabolism increased significantly between the 8- and 16-cell stages, but was accompanied by a much larger increase in glucose uptake. Consequently, the proportion of glucose uptake that was metabolized through the pentose-phosphate and Embden-Meyerhof pathways reached a minimum at those stages. From the compacted morula stage onward, the calculated uptake of [14C]glucose was only 25 to 33% of that calculated for [5-3H]glucose. This suggests that 66 to 75% of glucose carbon leaves the embryo, after metabolism to phosphoenolpyruvate, in some form other than CO2. Little or no glucose metabolism by the Krebs cycle could be detected at any stage. Both glutamine and pyruvate metabolism were relatively high at the 2- and 4-cell stages, declined to a minimum at the compacted morula stage and then increased with blastulation. Glutamine metabolism continued to increase with expansion and hatching of the blastocyst, but pyruvate metabolism did not. This suggest that, relative to the activity of the pathway from pyruvate to 2-oxoglutarate, the activity of the 2-oxoglutarate-to-oxaloacetate segment of the Krebs cycle is of increasing significance during expansion and hatching of the cattle blastocyst.

Cryopreservation of germ cells from bovine fetal ovaries.

Bovine fetuses at stages required for studies of female germ cells (primordial germ cells and oogonia) become available from the abattoir at unpredictable times. To alleviate this logistical problem, a procedure to cryopreserve these ovarian germ cells has been devised. Fetal ovarian cells were dispersed and suspended in 1.5 M dimethyl sulfoxide (DMSO) prepared in modified TCM 199 medium. The suspensions were aspirated into plastic semen straws, cooled, seeded to induce ice formation at -7 degrees C, and then cooled at 1 degree C min-1 to -70 degrees C before being plunged into liquid nitrogen at -196 degrees C for storage. The straws were thawed at a moderate rate of approximately 250 degrees C min-1, the DMSO was diluted 28-fold with culture medium, and then the cells were cultured for > 2 h before their viability was tested or they were used for nuclear transfer. No statistically significant difference in viability before and after cryopreservation was detected by vital staining with fluorescein diacetate (P > 0.05). When frozen-thawed germ cells were fused to cytoplasts, the cleavage rate of the resultant reconstructed embryos 44 h after fusion was 31%, although none developed into blastocysts. It is concluded that cryopreservation of bovine fetal ovarian germ cells is feasible and can play a major role in facilitating future experimentation.

Comparative aspects of equine embryonic development.

The developmental changes in the equine conceptus, its maternal environment and their interaction during the first 4 weeks following fertilization are reviewed. Attention is drawn to species-specific events to show why the horse is such a valuable model in which to study early pregnancy.

Reflections on the golden anniversary of the first embryo transfer to produce a calf.

In the belief that the vitality of a discipline benefits from a knowledge of its roots, events surrounding the first embryo transfer to produce a live calf (by Willett and his colleagues in Wisconsin fifty years ago) have been reviewed. That first success is discussed in the context of those times (including a brief review of the first international conference on embryo transfer, held in Texas in 1949) and in relation to subsequent events that led to the founding of the International Embryo Transfer Society in 1974.

Transcervical collection of equine conceptuses between 10 and 16 days after ovulation.

To recover intact Day-10.5 to Day-16.5 equine conceptuses (Day 0 = ovulation), a rigid catheter was used for 131 collections from donor mares diagnosed pregnant by ultrasonography. A total of 139 conceptuses were recovered, comprising 124 singletons, six pairs of twins and one set of triplets. Of these, 120 (86%) were intact after the collection, 14 (10%) had collapsed, and in five cases (4%), collapsed trophoblastic membranes were surrounded by an intact capsule. The recovery rate of intact conceptuses ranged from 99% on Days 10.5 to 12.5 to 40% on Day 16.5. More uterine flushes per recovery were needed to collect conceptuses on Day 14.5 than on Days 10.5 and 11.5 (x +/- SEM : 3.1 +/- 0.5 vs 1.4 +/- 0.1 and 1.3 +/- 0.2 flushes, respectively, P<0.05), and the total volume of flushing medium used was greater on Day 14.5 than on Days 10.5, 11.5 and 12.5 (1040 +/- 193 vs 406 +/- 49, 396 +/- 48 and 499 +/- 59 ml, respectively, P<0.01). Seventy of the 100 mares inseminated at the first estrus following embryo collection became pregnant, indicating that the technique used had no major effect on subsequent fertility.

In vitro fertilization and cleavage of in vivo matured bovine oocytes.

The effect of the interval between onset of estrus and oocyte collection on in vitro fertilization (IVF) rates has been investigated. The oocytes were surgically collected 6-18 h (Group I), 19-24 h (Group II), 25-29 h (Group III) and 30-36 h (Group IV) after the beginning of estrus. Recognizable stages of nuclear maturation were identified in 54.9% of the oocytes used for IVF (5.9% at germinal vesicle, 31.4% at metaphase I, 17.6% at metaphase II); the other 45.1% were degenerate. Considerable between- and within-cow variation in oocyte morphology, oocyte maturation and IVF results was observed. The overall fertilization and cleavage rates (to four-cell stages) were 26.5 and 6.0%, respectively. The fertilization rate increased as the interval between onset of estrus and collection increased and was optimal 30-36 h after onset. Thus, onset of estrus proved an effective means of timing oocyte collection for IVF.

Cellular composition and viability of demi- and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro.

The cellular composition and viability of intact, IVP embryos were compared with those of demi- and quarter-embryos produced by bisection of IVP morulae and blastocysts. Embryos were produced by established techniques from oocytes harvested from slaughterhouse ovaries. In Experiment 1, morulae at Day 6 or blastocysts at Day 7 were bisected on an inverted microscope using a microsurgical steel blade. Demi-embryos were then cultured without a zona pellucida until Day 8, when they were morphologically assessed for quality (viability). A higher proportion of demi-embryos made from blastocysts than from morulae were classified as viable (381/420, 91% vs 164/267, 61%; P < 0.001). In Experiment 2, only Day 7 blastocysts were bisected, and some of the resulting demi-embryos were bisected a second time 24 h later to produce quarter-embryos. The remaining demi-embryos, the quarter-embryos, and control intact embryos were cultured until Day 9, at which time they were assessed for quality and subjected to immunosurgery and differential staining to count inner cell mass (ICM) and trophectoderm cells. A higher proportion of demi-embryos than quarter-embryos was classified as viable (408/459, 89% vs 223/319, 70%, respectively; P < 0.001). Total cell numbers decreased with successive bisections, but the proportion of surviving cells found in the ICM was significantly (P < 0.05) higher in the best quality demi- and quarter-embryos (35 and 32%, respectively) than in the controls (22%). Transfer of all 12 quarter-embryos derived from 3 blastocysts, in pairs, into 6 recipient heifers resulted in 2 pregnancies, each with a single viable fetus at 90 d of gestation. The fetuses originated from 2 different blastocysts. The results suggest that bisection of intact IVP embryos into demi-embryos and bisection of those into quarter-embryos can increase the number of transferable embryos by as much as 178 and 235%, respectively.

Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro.

Various times of in vitro maturation of oocytes, and three methods of separating spermatozoa from frozen-thawed semen (Percoll density-gradient centrifugation, swim-up, and glass-wool filtration), were compared for their effects on goat embryo production in vitro. Cumulus-oocyte-complexes (COCs) from abattoir ovaries were matured in M199 supplemented with 10% fetal calf serum and hormones. In Experiment 1, COCs were fixed at 4 h intervals from 0 to 27 h of culture to assess oocyte nuclear maturation. A higher proportion cultured for 27 h than for 24 h were in Metaphase II (27/37, 73% vs. 18/33, 55%, P < 0.05). In Experiment 2, the effects of separation methods on total numbers and numbers of membrane-intact spermatozoa, and the acrosome reaction were compared. Total numbers after Percoll density-gradient centrifugation were approximately 4 times higher than after swim-up and approximately 2 times higher than after glass-wool filtration (P < 0.001). Progression of the acrosome reaction was not affected differentially. In Experiments 3 and 4, after 27 h of culture the COCs were inseminated with sperm isolated by the three methods. In Experiment 3, presumptive zygotes were examined for pronucleus (PN) formation at 6, 12, 18 and 24 h post-insemination. At 12 h, male PN formation rate from Percoll-treated spermatozoa was higher than from sperm subjected to swim-up and glass-wool treatments (20/37, 54% vs. 6/37, 16% and 6/38, 16%, respectively; P < 0.001). In Experiment 4, embryos were compared for cleavage at 48 h and development into blastocysts, hatching rates and cell number at 192 h. The rates of cleavage and blastocyst formation in the Percoll-treated group were higher (P < 0.05) than in the swim-up and glass-wool groups (62% and 18% vs. 50% and 11%, and 45% and 8%, respectively). Similarly, the mean cell number in the Percoll group was higher (P < 0.05) than in the swim-up and glass-wool groups (167 +/- 5 vs. 149 +/- 4 and 126 +/- 4, respectively). We conclude that Percoll density-gradient centrifugation is superior to the other two methods for separating goat spermatozoa from frozen-thawed semen in preparation for IVF.

The sex ratios of bovine embryos produced in vivo and in vitro.

The male:female ratio of developing bovine embryos produced and allowed to develop in vitro and in vivo was determined retrospectively from the cytogenetic analysis of 804 embyos. The overall male:female ratio of the 307 (38%) embryos that could be sexed was 162 (52.8%):145 (47.2%) and did not differ (P>0.05) from the expected 1:1 ratio. Among premorula stage embryos produced in vivo (n = 66) and in vitro (n = 30), the ratios were 1.2:1 and 0.76:1, respectively. Among morulae and blastocysts produced in vivo (n = 74), produced and cultured in vitro (n = 106, and produced in vitro and cultured in vivo (n = 31), the ratios were 1.11:1, 1.3:1 and 0.94:1, respectively, none of which differed significantly from 1:1. There was no difference (P>0.05) in the number of cells or mitotic index between male and female morulae and blastocyst, respectively.

Application of in vitro fertilisation techniques to obtain calves from valuable cows after slaughter.

The ovaries of two infertile cows of high breeding value were recovered after slaughter, and a total of 222 oocytes were obtained. Of these, 156 were classified as of good or fair quality and were subjected to in vitro maturation, in vitro fertilisation (using frozen semen from three bulls of high breeding value) and in vitro culture procedures. After eight days, 27 embryos were obtained, of which 13 were transferred fresh, and 14 were frozen. Three recipients of fresh embryos became pregnant; two calved and one aborted at four months. One of eight recipients of frozen-thawed embryos became pregnant but aborted at three months.

Production of sexed calves from frozen-thawed embryos.

Three experiments have been conducted with the aim of producing calves from frozen, sexed embryos by combining embryo splitting and cytogenetic methods. In the first experiment, the efficacy of the bisection technique was assessed by transcervical transfer of 10 monozygotic pairs of half embryos to 10 synchronised heifers. Thirteen calves were produced, including four sets of identical twins. In the second experiment, one of the halves of each of eight split embryos was transferred while the other was processed for sexing by identification of the sex chromosomes. In the third experiment, one of the halves from each of 28 embryos was frozen while the other half was used for sexing. Eleven of the 16 which were sexed have been transferred with the production of three calves of the predicted sex. The overall sexing rate was 60 per cent and the calving rate following transfer of sexed embryos was 60 per cent and 23 per cent for fresh and frozen halves respectively.

Production of four identical calves by the separation of blastomeres from an in vitro derived four-cell embryo.

The blastomeres of two in vitro derived four-cell embryos were separated and transferred individually into surrogate zonae pellucidae, then co-cultured with bovine oviductal epithelial cells for five days until blastulation. Pairs of the quarter blastocysts were co-transferred with trophoblastic vesicles into each of four synchronised Holstein heifers, three of which were diagnosed pregnant at 28 days gestation, carrying twin fetuses. Four genetically identical bull calves were delivered by elective caesarean section at term pregnancy. One pregnancy was terminated at 56 days.

Transfer of embryos from bovine leukaemia virus-infected cattle to uninfected recipients: preliminary results.

One hundred and fifty-one, day 6 or 7, embryos collected from cattle infected with bovine leukaemia virus (BLV) were transferred to uninfected recipients. Thirty-two pregnancies resulted. Two animals aborted at seven months. Three sets of twins and one single calf were still-born. The remaining 26 pregnancies produced 27 live calves which were raised to six months of age. All of the recipients, pregnant and non-pregnant, and all of the calves remained serologically negative for antibodies to BLV-glycoprotein antigen.

Transcervical embryo transfer in horses: an application in an equestrian teaching center.

Embryo transfer was used in an equestrian teaching center in order to produce as many foals as possible from their preferred mares during a single breeding season. Embryo collection by uterine lavage was attempted in five donor mares on 25 occasions 6.5 days after ovulation. Sixteen of the collection attempts (64%) yielded a total of 17 blastocysts. Of these 17 embryos, 13 were immediately transferred transcervically into recipient mares that had ovulated within two days of the time of ovulation in the donors, three were frozen for later transfer, and one was lost. Eight of the freshly transferred embryos (61%) developed and were detected on ultrasonography on day 11.5; five of these continued to develop normally and gave rise to healthy foals (38%), but three were lost at 14.5, 22.5 and 24.5 days gestation. Two of the frozen embryos were judged viable when thawed the following year and produced one additional pregnancy after transcervical transfer. Thus the five donor mares have produced five foals and a sixth 90-day pregnancy(1) with only a three-month interruption of their use for competition and teaching.(1)While this paper was in press, the sixth pregnancy also terminated in the production of a healthy foal.

Permeability of the equine embryonic capsule to ethylene glycol and glycerol in vitro.

Poor survival of cryopreservation by equine expanded blastocysts may involve low penetration of the embryonic capsule by cryoprotective agents (CPAs). This study characterized the permeation and accumulation rates of the CPAs ethylene glycol (EG) and glycerol (GLY) across isolated capsule in vitro, using a dual-chambered Valia-Chien permeation apparatus. Pieces of Days 14 to 18 ± 1 capsules separated media in the "donor" chamber containing either 1.5 M EG (n = 6), 0.74 M EG (n = 5), 0.87 M GLY (n = 7), or 0.15 M NaCl (saline, SAL) (n = 6), from the "recipient" chamber. Concentrations of CPA, determined by gas chromatography, allowed calculation of the capsule's apparent permeability (P(app)) to those CPAs. Permeation of capsule by 1.5 M EG was significantly more rapid than by 0.87 M GLY, or 0.74 M EG; permeation by both CPAs was significantly slower than by SAL. Accumulation of CPA in the recipient chamber depended more on initial donor chamber concentration, rather than type, of CPA. Accumulation rates for CPAs and SAL were linear only when capsule was present, demonstrating that their permeation through capsule was more complex than simple diffusion. Successful cryopreservation of equine expanded blastocysts has been previously linked to lengths of step-wise exposures to CPAs. Based on the present results, we inferred that alternative CPAs, more capable of permeating the capsule, or alternative methods of ensuring CPA entry into the cells, may also be required.