Osmotic behavior of in vitro produced bovine blastocysts in cryoprotectant solutions as a potential predictive test of survival

The osmotic behavior of bovine blastocysts produced in vitro was filmed during exposure to and dilution of cryoprotectant solutions used for vitrification. The relationship between the changes in the diameter of embryos and their subsequent survival was assessed. Embryos collected on Day 6 and Day 7 postinsemination were exposed to 10% glycerol (GLY) for 5 min, 10% GLY + 20% ethylene glycol (EG) for 5 min, and 25% Gly + 25% EG for 30 s, before dilution in 0.85 M galactose and finally in embryo transfer freezing medium (ETF). Embryos that had a higher probability of survival behaved as perfect osmometers, shrinking, reexpanding, or swelling according to an identical pattern, whereas embryos that deviated from this standard usually did not survive. The initial embryo diameter, duration of shrinkage and expansion in 10% glycerol, duration of reexpansion in ETF, and final embryo diameter were clearly predictive of the ability to hatch after culture in vitro. On a given day postinsemination, larger blastocysts were more likely than smaller blastocysts to survive and hatch after exposure to cryoprotectants with or without vitrification.     

Ontogenic corticosteroidogenesis of the domestic fowl: response of isolated adrenocortical cells

Ontogenic adrenocortical function of the domestic was investigated using adrenocortical cells isolated from embryonic chicks (18, 19, 20, and 21 days old) and male and female posthatch birds (1 day, 1 week, and 3 weeks old). Production of the predominant corticosteroids secreted by the chicken adrenal gland, corticosterone, cortisol, and aldosterone, was measured by radioimmunoassay after 2-hr incubation of cells with or without steroidogenic agents. Approaching hatch, basal and maximal ACTH-(1-24) (ACTH)-induced corticosteroid production increased steadily and peaked around 1 day posthatch (5-18 times and 3-9 times, respectively, the production values at 18 days embryonic life). Thereafter, corticosteroid production values decreased steadily to 3 weeks posthatch. Corticosterone predominated over the ages studied: Maximal ACTH-induced corticosterone production averaged 52 and 115 times the production values of aldosterone and cortisol, respectively. In addition, maximal ACTH-induced aldosterone production was roughly 2.2 times greater than cortisol production over the ages studied except for a short-lived, disproportionately greater aldosterone production at 1 day posthatch. In addition to perihatch and age-related differences in cellular corticosteroid production, there were also differences in cellular sensitivity to steroidogenic agents as indicated by the differences in half-maximal steroidogenic concentration values (ED50 values) of the steroidogenic agents. Sensitivity to ACTH increased 2.7 times from Day 18 of embryonic life to 1 day posthatch and then decreased steadily to 3 weeks posthatch. In addition, sensitivity to 8-bromo-cAMP (8-Br-cAMP) increased abruptly at 1 day posthatch (nearly 3 times) but then remained constant thereafter. However, a consistent change in cellular sensitivity to 25-hydroxycholesterol was not observed until 3 weeks posthatch (an increase in sensitivity of 3 times that at Day 18 of embryonic life). These data of cellular sensitivity suggest that there were distinct development and maturational alterations in the cellular loci at which ACTH, 8-Br-cAMP, and 25-hydroxycholesterol acted. Thus, during the transition from embryonic to postembryonic life of the domestic fowl, there are alterations in adrenocortical cell steroidogenic capacity and in the function of some cellular loci comprising the corticosteroidogenic pathway.     

Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo

The viability of embryos before flushing from donor mares (n = 5) and after transfer to recipient mares (n = 7) was monitored in mare serum by detecting early pregnancy factor (EPF) using the rosette inhibition test (RIT). The EPF activity was measured in donor mares before and after natural mating at natural estrus; after ovulation on Days 2, 5 and 8; and after embryo flushing (Day 8) on Days 8, 9, 10 and 13 after ovulation. The collected embryos were transferred immediately after flushing. The EPF activity in recipient mares were measured on the day of transfer and after embryo transfer on Days 1, 2, 3 and 5. Pregnancy was confirmed on Day 12 to 14 after embryo transfer. The mean EPF activity of donor mares was increased to the pregnant level (> an RI titer score of 10) on Day 2 after ovulation. Two days after flushing the embryos, the EPF activity of donor mares had decreased to the nonpregnant level. Among the 7 recipient mares, 3 mares were diagnosed pregnant on Day 12 after embryo transfer with ultrasound. The EPF activity of the pregnant recipient mares was increased above the minimum level observed in pregnant mares on Days 2 to 3 after transfer. However, among the nonpregnant recipient mares after embryo transfer, the EPF activity of 3 mares remained at the pregnant level only 2 to 3 d and then declined to the nonpregnant level. In one recipient mare, EPF activity did not reach the pregnant level throughout the sample collection. The results of this study indicated that equine EPF can be detected in serum of pregnant mares as early as Day 2 after ovulation. From our observation, we conclude that the measurement of EPF activity is useful for monitoring the in vivo viability of equine embryos and early detection of embryonic death.     

Corticosterone stimulates hatching of late-term tree lizard embryos

The regulation of hatching in oviparous animals is important for successful reproduction and survival, but is poorly understood. We unexpectedly found that RU-486, a progesterone and glucocorticoid antagonist, interferes with hatching of viable tree lizard (Urosaurus ornatus) embryos in a dose-dependent manner and hypothesized that embryonic glucocorticoids regulate hatching. To test this hypothesis, we treated eggs with corticosterone (CORT) or vehicle on Day 30 (85%) of incubation, left other eggs untreated, and observed relative hatch order and hatch time. In one study, the CORT egg hatched first in 9 of 11 clutches. In a second study, the CORT egg hatched first in 9 of 12 clutches, before vehicle-treated eggs in 10 of 12 clutches, and before untreated eggs in 7 of 9 clutches. On average, CORT eggs hatched 18.2 h before vehicle-treated eggs and 11.6 h before untreated eggs. Thus, CORT accelerates hatching of near-term embryos and RU-486 appears to block this effect. CORT may mobilize energy substrates that fuel hatching and/or accelerate lung development, and may provide a mechanism by which stressed embryos escape environmental stressors.     

Luteotrophic influence of early bovine embryos and the relationship between plasma progesterone concentrations and embryo survival

This study was carried out to evaluate the luteotrophic influence of early (before Day 7 as well as after Day 7; Day 0=estrus) bovine embryos and the relationship between plasma progesterone (P4) concentrations and embryo survival. Virgin Holstein dairy heifers (n=325) from a single herd were randomly allocated to be nonbred, bred by artificial insemination (AI) or by embryo transfer (ET). Bred heifers were either treated with 1500 IU human chorionic gonadotrophin (hCG) on Day 7 of the estrous cycle or received no hCG treatment. Plasma P4 concentrations on Days 0, 5, 7, 10, 13, 15, 17, 19 and 21 were similar in pregnant AI- and ET-bred heifers and, this was observed in both hCG-treated and untreated females. Nonbred, AI- and ET-bred nonpregnant heifers (both hCG-treated and untreated) presented similar plasma P4 concentrations. Plasma P4 concentrations of pregnant heifers significantly deviated from those of nonpregnant and nonbred heifers on Day 17. In hCG-treated heifers, plasma P4 concentrations and Day 28 pregnancy rate were significantly higher in females with an induced accessory corpus luteum (CL) than in those females without an induced accessory CL. Treatment with hCG, although inducing the formation of accessory CL and significantly increasing plasma P4 concentrations had no significant effect on Day 28 pregnancy rate. In conclusion, this study does not support the existence of any peripherally detectable luteotrophic influence from early embryos (Days 5-7). Plasma P4 was only significantly related to embryo survival on Day 17, the time of expected onset of luteolysis.     

In vitro development of bovine embryos cultured with activin A

The aim of this study was to investigate the effects of activin A on development, differential cell counts and apoptosis/necrosis rates of bovine embryos produced in vitro. Presumptive zygotes were cultured up to Day 8 in synthetic oviduct fluid containing aminoacids, citrate, myo-inositol and BSA. In Experiment 1, activin (10 ng mL⁻¹) was added: 1/from Day 1 to Day 3; 2/from Day 1 to Day 8; 3/from Day 3 to Day 8; or 4/absent (control). In Experiment 2, 10 ng mL⁻¹ activin were added either before (Day 3 to Day 5) or after (Day 5 to Day 8) the early morula stage. In Experiment 1, activin during the first 72 h of culture reduced Day 3 cleavage, 5-8 cell rates and blastocyst development, while hatching rates increased. No changes were observed within differential cell counts. In experiment 2, activin improved blastocyst development after, and had no effect before, the Day 5 morula stage. However, trophectoderm (TE) cell numbers decreased with activin both before and after the Day 5 morula stage, suggesting that activin inhibits TE differentiation. The presence of activin during the whole culture had no effect on TUNEL positive cells, but when added at shorter periods activin increased apoptotic rates. Effects of activin during in vitro bovine embryo development, depends on timing of its addition to the culture medium.     

Survival of day-4 embryos from young, normal mares and aged, subfertile mares after transfer to normal recipient mares

The estimated embryonic loss rate between Days 4 and 14 after ovulation for young, normal mares (9%) was significantly lower (P less than 0.01) than the estimated embryonic loss rate for aged subfertile mares (62%). Fertilization rates, which were based on the recovery of embryos at Day 4 after ovulation, were 96% and 81% (P less than 0.1) for normal and subfertile mares, respectively. Day-4 embryos were collected from the oviducts of normal and subfertile donors mares. These embryos were transferred to the uteri of synchronized, normal recipient mares to test the hypothesis that the high incidence of embryonic loss in subfertile mares was related to embryonic defects. The hypothesis was supported because embryo survival rates were significantly higher (P less than 0.05) for Day-4 embryos from normal compared to subfertile mares. These defects may have been intrinsic to the embryo or might have arisen due to the influence of the oviducal environment before Day 4 after ovulation.     

Precocious development of UDP-glucuronyltransferase activity in chick-embryo liver after administration of adrenocorticotropic hormone and of certain 11beta-hydroxy corticosteroids.

1. Precocious development of UDP-glucuronyltransferase (EC 2.4.1.17) and of glucuronidation by endogenous compounds of known chemical composition is reported for the first time. 2. This development occurs precociously in chick-embryo liver after administration to the egg of mammalian adrenocorticotropic hormone, of Synacthen (a synthetic compound possessing adrenocorticotropic activity), or of certain corticosteroids possessing a hydroxy or an oxo group at C-11. 3. Corticosterone-dependent transferase development parallels the rise of infused corticosterone in plasma, but does not require the presence of embryo pituitary in ovo, and is demonstrable in embryo liver explants in vitro. 4. Competence of embryo liver transferase to respond to corticosterone (or dexamethasone) begins over days 13-14, the time of competence to respond to grafted pituitary gland. 5. The transferase appearing after treatment with corticosterone or adrenocorticotropic hormone, like that appearing after pituitary grafting or on natural development and unlike that from the untreated embryo, is markedly activated by membrane-perturbation procedures, suggesting it appears through induction, not activation. 6. Thyroxine and tri-iodothyronine accelerate transferase development after treatment with adrenocorticotropic hormone or corticosteroid to the rate seen after pituitary grafting. 7. A wide range of other hormones and steroids did not obviously influence transferase development in this system. 8. We suggest that grafted pituitary gland evokes precocious transferase development in embryo liver through production of adrenocorticotropic hormone and hence of the active corticosteroids; thyrotropin and thyroxine hasten the process. The role of this mechanism in the natural development of UDP-glucuronyltransferase is discussed.     

Total unconjugated oestrogen and progesterone concentrations in peripheral blood during the oestrous cycle of the dog.

From the beginning of pro-oestrus to the end of metoestrus, daily peripheral blood samples were withdrawn from six bitches. Further samples were obtained during anoestrus. Oestrogen values rose from the onset of pro-oestrus to attain a peak value of 25-3 +/- 4-8 pg/ml on Day 1 of oestrus. Progesterone concentrations began to rise 2 days before the oestrogen peak, and reached their highest levels of 18-9 +/- 1-0 ng/ml 16 days after the end of oestrus. After oestrus, an oestrogen rise was detected which reached a peak at Day 18 of metoestrus. The duration of oestrus was unrelated to the length of time that oestrogen levels were raised, or the maximum values were attained. No significant oestrogen levels were detected during anoestrus.     

Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: Can supplementary progesterone rescue small embryos?

Conceptus size on Day 14 after multiple embryo transfer of Day 7 in vitro–produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development after the transfer of Day 7 blastocysts containing a low total cell number. The estrous cycles of crossbred beef heifers were synchronized using an 8-day progesterone (P4)–releasing intravaginal device (PRID) with the administration of a prostaglandin F_2α analog on the day before device removal. Only those heifers recorded in standing estrus (Day 0) were used. Heifers were randomly assigned to one of four treatment groups: (1) control: large blastocysts (high total cell number), (2) control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU human chorionic gonadotropin (hCG) on Day 2 after estrus, or (4) small blastocysts plus insertion of a vaginal P4 insert (PRID, 1.55 g P4) between Days 3 and 5 after estrus. In vitro–produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer), and conceptuses were recovered at slaughter on Day 14. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analyzed using the PROC MIXED procedure of SAS. Total cell number on Day 7 was significantly lower in small versus large blastocysts (72.4 ± 3.93 vs. 144.8 ± 3.90, P < 0.05). Conceptus recovery rate was 53.8% overall (140 of 260) and was highest in the large blastocyst group (68.3%, 41 of 60) compared with the other groups (45.7%–55.0%). Concentrations of serum P4 were similar in the two unmanipulated recipient groups but were significantly elevated (P < 0.05) by Day 8 in the hCG-treated heifers and on Days 4 and 5 in the PRID group (P < 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm; P < 0.05), whereas insertion of a PRID from Days 3 to 5 increased conceptus length approximately fivefold (13.09 ± 2.11 mm; P < 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of “rescuing” poor-quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition.     

Comparison of two manipulation methods to produce in vitro fertilized, biopsied and vitrified bovine embryos

The objective of this study was to compare the overall efficiency, measured by in vitro embryonic survival, and practical value of bovine in vitro embryo production, biopsy, vitrification, and direct transfer technology using 2 different manipulation methods for biopsy. Slaughterhouse-derived oocytes were matured in vitro, fertilized (Day 0) with frozen-thawed, Percoll-separated spermatozoa and cultured on a granulosa cell monolayer. In Experiment 1, one or two blastomeres were expelled from Day 4 embryos by mechanical force through a hole made by partial zona dissection. Using a darning needle hole system for individual culture of biopsied embryos from Day 4 to Day 7.5, the blastocyst per oocyte rate was 50%, and 76% of the blastocysts survived subsequent vitrification and direct in-straw rehydration. Attempts to increase the cell number of the biopsies by further in vitro culture were unsuccessful. In Experiment 2, Day 7 and Day 8 embryos were manually biopsied before or after vitrification. When biopsy was performed before vitrification, 98% of the embryos survived manipulation, and 86% of these re-expanded after vitrification and in-straw dilution. Biopsy after vitrification was less efficient, since only 69% of the embryos survived both processes. The cumulative efficiency of embryo production, Day 7.5 biopsy and vitrification--in-straw direct rehydration was lower (P < 0.001) than that of Day 4 biopsy and Day 7.5 vitrification (29 vs 38%, respectively). However, a Day 7.5 biopsy may have the more practical application since the size of the biopsy is larger and the process is not as time-consuming as the long-term individual culture of the biopsied embryos.     

The effect of recipient age on the success of embryo transfer in mice

Eight-hundred-sixteen morulae and blastocysts were transferred to the uteri of 102 recipients of various ages on Day 3 or 4 of pseudopregnancy (Day 1 is the day of the vaginal plug). Day 3 recipients had significantly higher pregnancy rates and embryo survival rates than Day 4 recipients. Recipient age had little effect on pregnancy rates, but had a significant effect on embryo survival in Day 3 recipients. Day 3 recipients of 11-13 weeks of age had the highest pregnancy rate (100%) and embryo survival (75%). The results suggest that recipient age should be considered an important factor in embryo transfer experiments.     

Transuterine embryo migration in recipient cattle

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

Studies on avian erythrocyte metabolism: IX. Relationship of changing organic phosphate composition to whole blood oxygen affinity during development of the ostrich (Struthio camelus camelus)

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in erythrocytes (RBC) of the 37-day ostrich embryo at a concentration of 3.7 μmole/ml RBC, representing 23.5% of the cell phosphate. (2) Inositol tetraphosphate (inositol-P₄) is absent in red cells of the 37-day embryo, appears in the RBC about 63 days after hatch, and thereafter gradually accumulates to a level of 2.8 μmole/ml RBC in the adult bird, representing 30–35% of the cell phosphate. (3) Inositol pentaphosphate (inositol-P₅) is present in the erythrocytes of the 37-day embryo at a concentration of 0.8 μmole/ml RBC, reaches a peak level of 2.9 μmole/ml RBC by Day 63 after hatch, and thereafter declines to a level of 1.1 μmole/ml RBC in the adult bird. (4) The crossover time where the molar concentrations of inositol-P₅ and inositol-P₄ are equal in the erythrocyte appears to be about 150 days after hatch. (5) The p₅₀ of whole blood from the 37-day ostrich embryo, 5-day ostrich chick, and adult ostrich was 15.4, 31.8, and 24.9 Torr. The p₅₀ of whole blood immediately after hatch correlates best with an abrupt rise in ATP concentration but after 54 days posthatch correlates best with the appearance of increasing concentrations of inositol-P₄ and the decrease in concentrations of ATP and inositol-P₅. (6) The appearance of inositol-P₄ in the cells could be an adaptive mechanism for regulating oxygen supply by switching to a modulator which maintains a higher oxygen affinity than would a predominance of inositol-P₅.     

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.     

自分泌生长因子对早期胚胎体外发育的影响（简报）

Two-cell-stage embryos were flushed from the oviducts on Day 2. Zygotes were collected from oviducts on Day 1 (Fertilization In Situ, ISF) or derived from fertilization in vitro (IVF). 2-cell embryos had a high rate of blastocyst development to each embryo concentration from 1 embryo/microliter to 1 embryo/1000 microliters. The zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (P < 0.001), with approximately 82.5% of ISF zygotes developing to blastocysts at highest concentration but only 22.3% at the lowest. For IVF zygotes the corresponding results were 46.3% and 5.2%. The number of cells in each blastocyst from 2-cell embryos was significantly higher than that from ISF and IVF group. The media supplementing Platelet-activating factor (PAF) caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentration of 1 embryo/10 microliters (10 ng/ml) and 1 embryo/100 microliters (100 ng/ml). Insulin-like growth factor (IGF) (10 ng/ml) also stimulated development of IVF zygotes when they were cultured at the concentration of 1 embryo/10 microliters. Epidermal growth factor (EGF) was no effect over range of 1-1000 ng/ml to embryo development. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The PAF, IGF-I partially compensate the effects of low embryo concentration during culture and play important roles as autocrine embryotrophic factors.     

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.     

Lectin activity from embryonic chick brain,heart, and liver: Changes with development

Extracts of embryonic chick brain, heart, and liver agglutinate glutaraldehyde-fixed trypsinized or pronase-treated rabbit erythrocytes. Agglutination activity of extracts from each organ was inhibited by a number of saccharides. Lactose was the most potent saccharide inhibitor of those tested. The specific agglutination activity of the extracts from each of the organs studied changed with development of the embryo. In general, specific agglutination activity declined later in embryogenesis, and after hatching. However, the pattern of developmental change differed for each of the organs tested. Liver was unusual in that, after hatching, agglutination activity rose again; and the agglutinin found at this time was apparently different from that found in the embryo.     

Uterine luminal proteins and estrogens in gilts on a normal nutritional plane during the estrous cycle and on a normal or high nutritional plane during early pregnancy

In gilts, a high plane of nutrition during early pregnancy often results in increased embryo mortality, possibly related to changes in embryo-uterine asynchrony at a critical stage of pregnancy (around Day 11). Therefore, in the present study, uterine luminal proteins and estrogens were studied between Days 5 and 16 after the onset of estrus in gilts on either a normal (2.5 kg/d, cyclic and pregnant gilts) or a high (4.0 kg/d, pregnant gilts only) feeding level. Conceptus recovery rate between Days 5 and 12 was not affected by the feeding level during early pregnancy, neither were systemic progesterone levels. Between Days 9 and 11, dramatic changes took place in the protein composition of the uterine luminal 10kD+ proteins, shifting from most (90%) of the acidic proteins at Day 5 and 7 to approximately 50% at Day 11/12, especially due to an increase in basic proteins with an iso-electrical point of more than 8. This shift occurred most rapidly for the pregnant gilts at the high feeding level and least rapidly in the cyclic gilts, resulting in significant differences in the relative amount of acidic proteins at Day 10 and 11 after the onset of estrus (P < 0.05). Similarly, levels of estrogens in the uterine flushings at Days 10, 11 and 12 were always highest for the pregnant gilts on the high feeding level and were always lowest in the cyclic gilts (P < 0.05); pregnant gilts on the normal feeding level showed intermediate estrogen levels. The fact that gilts on a high feeding level during early pregnancy show more rapid changes in the uterine luminal protein composition and embryonic estrogen production seems to suggest that the rate of these changes may be related to embryo survival.     

The influence of time of insemination relative to time of ovulation on farrowing frequency and litter size in sows, as investigated by ultrasonography

The objective of this experiment was to identify the optimal time of insemination relative to the time of ovulation, based on ultrasonographic detection of embryonic survival at 10 days after ovulation, number of sows farrowing, and litter size. Furthermore, the possible value of the interval from weaning to onset of estrus for prediction of the time of ovulation was examined. Crossbred sows (n = 143) that had farrowed 2 to 9 litters were weaned (Day 0) and observed for estrus every 8 h from Day 3 until end of estrus. Ultrasonography was performed every 6 h, from 12 h after onset of estrus until ovulation had been observed. The sows were inseminated once at various time intervals from ovulation. At Day 16, 25 of the sows were slaughtered and their uteri were flushed for embryos. In the remaining sows, the number of viable and dead piglets and mummified fetuses per sow was recorded at farrowing, with the sum of the 3 constituting the total number of piglets born per sow. The highest number of embryos recovered per sow was found after insemination during the interval from 24 h before to 4 h after ovulation. The lowest frequency of non-pregnant sows and the highest total number of piglets born per sow were found after insemination from 28 h before to 4 h after ovulation. Consequently, the optimal time for insemination was found to be in the interval 28 h before to 4 h after ovulation. The interval from weaning to onset of estrus and from onset of estrus to ovulation were negatively correlated, allowing a rough prediction of the time of ovulation from the interval from weaning to onset of estrus.