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.     

High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1-2 propanediol as cryoprotectant

A method for obtaining a high survival rate of frozen-thawed mouse embryos is presented. Eight-cell mouse embryos were frozen inside small plastic straws in the presence of 1-2 propanediol and stored at -196 C. After thawing, the embryos were diluted for only 5 min in a 1.0 M sucrose solution to remove the 1-2 propanediol from the cells. At high rate of thawing (is equivalent to 2500 C/min) more than 88% of the embryos survived in vitro to the blastocyst stage provided that the dilution of propanediol was performed rapidly during thawing. At a lower rate of thawing (is equivalent to 300 C/min), survival tended to be higher (94.7%) when dilution was done 5 min after thawing. When the frozen-thawed embryos were transferred to the oviducts of day 1 pseudopregnant recipients either directly after the dilution of 1-2 propanediol or after 24 or 48 hr of culture, a high proportion of them (65.9%) develop normally to viable fetuses.     

应用乙二醇冷冻小鼠胚胎：优化和简化程序的探索

提高解冻胚胎的发育能力和简化冷冻解冻程序是胚胎冷冻研究的两大永恒的主题。尽管乙二醇（EG）广泛用于家畜胚胎冷冻,但很少用于冷冻小鼠和人胚胎。为数很少的以EG慢冻小鼠或人胚胎的研究均采用较为复杂的人胚冷冻程序,未见简化程序和用EG冷冻小鼠桑椹胚的报道。采用简单的牛胚胎冷冻程序研究了发育时期、EG浓度、平衡方法、添加蔗糖以及解冻后脱除EG等对小鼠胚胎冻后发育能力的影响。结果显示：（1）致密晚期桑椹胚冻后体外培养囊胚发育率（81.92%±2.24%）和孵出率（68.56％±2.43%）显著（P＜0.05)高于4-细胞、8-细胞胚胎和致密早期桑椹胚胎;（2）1.8mol/L EG冷冻小鼠致密晚期桑椹胚的囊胚发育和孵出率显著高于其它浓度;（3）在EG中平衡10min的冻后囊胚发育显著好于平衡5、20或30min;（4）两步平衡冷冻胚胎的囊胚发育率和孵出率显著高于一步平衡;（5）用EG冷冻小鼠胚胎无需添加蔗糖;（6）解冻后可不脱除EG;（7）冻后发育的早期囊胚和囊胚细胞数明显少于体内发育胚胎。因此,用EG冷冻小鼠胚胎的最佳方案为：致密晚期桑椹胚用1.8mol/L EG不添加蔗糖、两步平衡15min、以简单的牛胚胎冷冻程序冷冻解冻、解冻后不脱除EG直接培养或移植。     

In vitro culture of goat morulae to blastocysts before freezing

The results of transfer of frozen-thawed caprine embryos that were collected either as blastocysts or morulae and cultured to the blastocyst stage prior to freezing were compared. After thawing, the embryos collected as blastocysts appeared to be of marginally better quality than those that had been cultured from morulae (89 vs 72% rated as good; P > 0.05). The transfer of 24 frozen-thawed embryos collected as blastocysts to 12 recipients resulted in a pregnancy rate of 83% (10/12) and an embryo survival rate of 67%. Corresponding results for frozen-thawed blastocysts that had been cultured from morulae and were transferred to 11 recipients were 54% (6/11) and 41%, respectively. Since an earlier investigation had shown that the transfer of frozen caprine morulae yields very poor results, in our laboratory all morulae are now cultured to the blastocyst stage before being cryopreserved.     

Beneficial effect of agar for the frozen storage of bisected embryos

The effect of agar embedding on the viability of intact and bisected goat embryos during freezing and thawing was examined. Blastocysts or hatched blatocysts were bisected into haves by micromanipulation. After embedding with or without agar, they were stored by deep freezing. After thawing, undamaged and some partially damaged embryos were transferred into uteri of recipients. Of 22 demi-embryos embedded with agar, only one was undamaged, but of 58 demi-embryos embedded with agar, 29 were undamaged. Although one set of monozygotic twins was obtained after the transfer of 15 sets of frozen-thawed bisected embryos, the pregnancy rate (47%) and the proportion of young (27%) were lower than those obtained by transfer of frozen-thawed, intact embryos (67 to 80% for pregnancy rate, 45 to 53% for the young).     

Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen-thawed two-cell mouse embryos

Physical and chemical alterations caused by the freezing and thawing and their effects on survivals/developments in vitro were investigated. Of a total of 452 two-cell mouse embryos, the overall survival rate of the frozen-thawed embryos was 76.1% (344/452). The blastocyst formation of the frozen-thawed embryos was 32.6% (44/136) compared to 74.5% (117/157) in the fresh embryos (P<0.05). The total number of cells in a blastocyst also decreased from 96.0 +/- 19.0 (n=26) in the fresh embryos to 42.0 +/- 11 .34 (n=30) in the frozen-thawed embryos (P<0.05). Fluorescence recovery after photobleaching (FRAP) measurement revealed about 5-fold decrease in the cell membrane fluidity with a characteristic time constant (tau) of 1.46 +/- 0.13 sec (n=5) in the frozen-thawed embryos as opposed to 0.28 +/- 0.04 sec (n=5) in the fresh embryos (P<0.05). The relative amount of H(2)O(2) in an embryo as quantified by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) showed 62.8 +/- 23.5 (n=24) and 34.2 +/- 14.5 (n=20) in the frozen-thawed embryos and in the fresh embryos, respectively (P<0.05). The distribution of actin filaments in the frozen-thawed embryos revealed an uneven distribution, particularly discontinuities at the "actin band," which contrasted to an even distribution shown in the fresh embryos. Mitochondrial staining by Rhodamine 123 showed that there was no significant difference between the two treatments in the number and in the distribution of viable mitochondria, but a marked aggregation was seen in the arrested embryos. No Annexin V binding was detected in two-cell or four-cell embryos while the binding was positive in the arrested embryos. The mitochondrial membrane potential measured by a membrane potential-sensitive fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol- carbocyanine iodide (JC-1) revealed a marked depolarization in the frozen-thawed embryos. Finally, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was employed to quantify the DNA fragmentation. In 75.0% cells of blastocysts (n=24) in the frozen-thawed embryos, the DNA fragmentation was detected as opposed to 37.0% in the fresh embryos (n=20) (P<0.05). Taken together, it is proposed that during the cryopreservation, two-cell mouse embryos are subjected to physical and chemical alterations, including destruction of the cell membrane integrity, redistribution of actin fibers, mitochondrial depolarizations, and increased reactive oxygen species (ROS) productions, which then may trigger the apoptotic cascade leading to a decrease in the survival rate and in the developmental rate of the embryos.     

Pregnancy rates in Holstein embryo transfer recipients: Effect of treatment with progesterone or clenbuterol and of natural versus induced cycles

Pregnancy rates were the same (59 and 50%; P > 0.05) in control recipients (n = 22) and recipients treated with 200 mg progesterone 4 h prior to nonsurgical transfer (n = 48) of previously frozen embryos. With small recipients and difficult transfer conditions, the same progesterone supplementation increased pregnancy rates (46 vs 21%; P < 0.05) after transfer of previously frozen embryos. The administration of the β₂-mimetic agent clenbuterol (4-amino- α [(tert. - butylamino) methyl] - 3,5,dichlorobenzyl-alcohol hydrochloride) 30 min prior to nonsurgical transfer resulted in pregnancy rates of 60.0% (n = 25) and 44.2% (n = 52) for Holstein heifers receiving fresh and previously frozen embryos, respectively. Pregnancy rates in untreated controls were 59.9% (n = 142) and 48% (n = 47) for fresh and frozen-thawed embryos. There was no effect (P > 0.05) of spontaneous versus prostaglandin-induced estrus on pregnancy rates for recipients of either fresh or previously frozen embryos.     

Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle.

The effects of a large number of factors on the pregnancy rates of fresh and frozen cattle embryos were examined over a period of years at several different locations. For fresh embryos, overall pregnancy rates were 68.3% (n=9023) and 77.1% (n=2650) at different locations and time periods. Frozen-thawed embryo pregnancy rates were 56.1% (n=3616) in The Netherlands and 58.4% (n=5297) and 68.7% (n=774) for two studies in the United States. Pregnancy rates of surgical versus nonsurgical transfers were very similar. There were no differences in the pregnancy rates of beef versus dairy embryos, but the pregnancy rate was higher in dairy and beef heifers and beef cows than in dairy cows. Although on-farm pregnancy rates in California were higher than in the northeast United States, there was no influence of season on pregnancy rate. Estrous asynchrony between plus and minus 24 h did not affect pregnancy rate for frozen-thawed or fresh embryos. Neither breed nor parity of recipients affected the influence of asynchrony on pregnancy rates. Embryo grade was a significant factor in pregnancy rate for both fresh and frozen-thawed embryos, but neither embryo stage nor age was a significant factor. Pregnancy rate was not affected by holding embryos after flushing for up to 3 h prior to freezing.     

Interaction between season and culture with insulin-like growth factor-1 on survival of in vitro produced embryos following transfer to lactating dairy cows

Culture of bovine embryos in the presence of insulin-like growth factor-1 (IGF-1) can increase pregnancy rates following transfer to heat-stressed, lactating dairy cows. The objective of the present experiment was to determine whether the effect of IGF-1 on post-transfer embryo survival was a general effect or one specific to heat stress. Lactating recipients (n=311) were synchronized for timed-embryo transfer at four locations. Embryos were produced in vitro and cultured with or without 100 ng/mL IGF-1. At Day 7 after anticipated ovulation (Day 0), a single embryo was randomly transferred to each recipient. Pregnancy was diagnosed at Day 21 by elevated plasma progesterone concentrations, at Days 27-32 by ultrasonography, and at Days 41-49 by transrectal palpation. Transfers were categorized into two seasons, hot or cool (based on the month of transfer). There was a tendency (P<0.09) for an interaction between embryo treatment and season for pregnancy rate at Day 21; this interaction was significant at Days 30 and 45 (P<0.02). Recipients receiving IGF-1 treated embryos had higher pregnancy rates in the hot season but not in the cool season. There was a similar interaction between embryo treatment and season for overall calving rate (P<0.05). There was also an interaction between season and treatment affecting pregnancy loss between Days 21 and 30; recipients that received IGF-1 treated embryos had less pregnancy loss during this time period in the hot season but not in the cool season. The overall proportion of male calves born was 77.5%. In conclusion, treatment of embryos with IGF-1 improved pregnancy and calving rates following the transfer of in vitro produced embryos into lactating recipients, but only under heat-stress conditions.     

Repeat superovulation, non-surgical embryo recovery, and surgical embryo transfer in transgenic dairy goats

We investigated the capability of repeat superovulation and non-surgical embryo retrieval, coupled with surgical embryo transfer, to expedite the production of transgenic progeny from transgenic founder dairy goat does. In addition, we compared embryo yields, number of embryos transferred per recipient, pregnancy rates, and offspring born during both the traditional (September-December) and non-traditional (January-May) breeding seasons. Although there were no significant differences, there were numerically more transferable embryos recovered per flush (3.5+/-0.9 vs. 2.4+/-0.9 embryos; mean+/-S.E.M.) and increases in both the proportion of recipients that were pregnant (83 vs. 69% pregnant) and offspring born from total embryos transferred (67 vs. 53% offspring) during the traditional versus the non-traditional breeding season. The transfer of one, two or three embryos did not significantly affect the proportion of pregnant recipients during either season. However, there was a difference (P<0.05) in the proportion of offspring produced for one versus two embryo transfers (89 vs. 44% offspring, respectively) during the non-traditional breeding season. Overall, 14 transgenic offspring were produced from 54 total offspring born, and the kidding interval was reduced to <3 months for six of the seven transgenic does. In summary, repeat superovulation and non-surgical embryo retrieval, coupled with surgical embryo transfer, expedited the production of progeny from transgenic founder does.     

Quick freezing of one-cell mouse embryos using ethylene glycol with sucrose

One-cell mouse embryos were frozen by direct plunging into liquid nitrogen (LN(2)) vapor after equilibration in 3 M ethylene glycol with 0.25 M sucrose (freezing medium) for 5 to 40 minutes. After thawing, the embryos were cultured in vitro and the effects of the equilibration period and dilution method were examined. No significant difference was observed in the in vitro survival of embryos when 0.5 or 1.0 M sucrose was used for the dilution of the cryoprotectant for each equilibration period. The highest survival rate (67.2%) was obtained when the embryos were equilibrated for 10 minutes, and the cryoprotectant diluted with either 0.5 or 1.0 M sucrose after thawing. Shorter (5 minutes) or prolonged (40 minutes) equilibration of embryos in the freezing medium yielded significantly lower survival rates. Dilution by direct transfer of the frozen-thawed embryos into PB1 resulted in lower survival rates than when 0.5 or 1.0 M sucrose was used. The in vitro development to the blastocyst stage of one-cell mouse embryos frozen after 10 minutes equilibration in the freezing medium and diluted after thawing in 0.5 M sucrose was significantly lower than the control (68.0 vs 92.7%). However, transfer of the blastocysts developing from frozen-thawed one-cell mouse embryos into the uterine horns of the recipients resulted in fetal development and implantation rates similar to the control.     

The use of embryo transfer to produce pregnancies in repeat-breeding dairy cattle

Repeat breeding is an important factor affecting economic success in dairy management. The objective of this study was to investigate the effectiveness of transfer of frozen-thawed IVF embryos in establishing pregnancy in repeat-breeding Holstein cattle. Cumulus oocyte complexes were collected by aspiration of 2-5 mm follicles from ovaries obtained at two local abattoirs. After IVF, days 7 and 8 blastocysts were frozen either in 1.5M ethylene glycol with 0.1M sucrose, or in 1.4M glycerol with 0.1M sucrose. Holstein recipients (122 heifers and 410 cows) included those that had not conceived after 3-21 inseminations. Embryos frozen in ethylene glycol were transferred directly, and embryos frozen in glycerol were transferred after dilution of the cryoprotectant in sucrose into recipients 7 or 8 days after estrus (without-AI group), or following AI (with-AI group). Pregnancy rates were compared by the Chi-square test. Significantly higher pregnancy rates were achieved by embryo transfer following AI (with-AI group) than by embryo transfer alone (without-AI group) in both heifers (49.2 and 29.5%, respectively) and cows (41.5 and 20.4%, respectively; P<0.05). Pregnancy rates were not significantly different between heifers and cows. However, pregnancy rate decreased as the number of inseminations prior to embryo transfer increased in the with-AI group, but not in the without-AI group. Therefore, transfer of frozen-thawed IVF embryos during the same cycle in which AI was done improved pregnancy rates in repeat-breeding Holstein heifers and cows, and suggested that embryo transfer is an alternative in the treatment of repeat breeding.     

Season affects characteristics of the pre-ovulatory LH surge and embryo viability in superovulated ewes

The aim of this study was to determine whether there are seasonal shifts in ovulatory response, and in the viability of ova recovered from superovulated ewes. Fifty mature ewes underwent a standard oestrous synchronisation (CIDR), superovulation (oFSH) and artificial insemination procedure during October (peak breeding season) and April (transition to anoestrus). In each month peripheral LH and progesterone concentrations were measured around the time of ovulation and embryos were recovered, graded and cryopreserved on day 6 after insemination. During the subsequent breeding season, grade 1 and 2 morulae and unexpanded blastocysts were thawed and transferred singly to synchronous recipients (October, n = 40; April, n = 40) or cultured in vitro for 18-20 h (October, n = 107; April, n = 98). Following culture, viable embryos were stained to count cell nuclei or assayed to measure their capacity for glucose metabolism ([3H]glucose) and protein synthesis ([35S]methionine). Peak LH concentrations were higher in October than in April (38.2 +/- 3.26 ng ml(-1) versus 25.7 +/- 1.99 ng ml(-1), respectively; P < 0.01) and the pre-ovulatory LH surge was advanced by approximately 3 h (P < 0.05). Progesterone concentrations at CIDR withdrawal were lower in October than in April (3.1 +/- 0.16 ng ml(-1) versus 4.3 +/- 0.19 ng ml(-1), respectively; P < 0.001) but were not different at embryo recovery. Season did not affect the numbers of corpora lutea per ewe or the numbers of ova recovered but the proportion of recovered ova that was unfertilised/degenerate was lower in October than in April (0.43 versus 0.58, respectively; P < 0.001). For embryos containing more than 16 cells, there was no effect of season on the median stage of development or morphological grade. The proportions of October and April embryos that established pregnancy following transfer to recipient ewes were 0.78 and 0.70 (not significantly different), and that were viable after in vitro culture were 0.66 and 0.37 (P < 0.05), respectively. Season did not affect the number of nuclei per viable embryo or the capacity for protein synthesis but the glucose uptake of October embryos was approximately double that of April embryos (3163+/-293.4 dpm versus 1550+/-358.9 dpm, respectively; P < 0.05). Results indicate that during the late compared to peak breeding season, there is an increased incidence of fertilisation failure as a possible consequence of seasonal shifts in LH secretion and (or) associated effects on follicular function. Frozen-thawed embryos produced at contrasting stages of the breeding season are equally viable in vivo but those produced during the late, as opposed to the peak breeding season have lower viability following in vitro culture.     

Non-surgical deep intra-uterine transfer of in vitro produced porcine embryos derived from sex-sorted frozen-thawed boar sperm

Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with unfrozen sperm, and after surgical insemination with sex-sorted frozen-thawed sperm. The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a pre-determined sex and that these embryos could be successfully non-surgically transferred. Oocytes were matured in vitro, fertilised with either unsorted or sex-sorted frozen-thawed sperm and cultured until the eight-cell stage. These embryos were then transferred to recipients (n = 7) non-surgically (n = 70 embryos per sow). Oocyte cleavage was similar between sex-sorted (1538/5044; 30.5%) and unsorted (216/756; 28.6%) frozen-thawed sperm, and PCR sex-determination of the embryos confirmed that they were of the predicted sex (n = 16). Delayed return to oestrus (>23 days) was observed in five recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound on Day 18 in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine ET protocols are required to enable development to term.     

Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo (Bubalus bubalis): a retrospective study

The objectives of this study were to determine the pregnancy rate and factors affecting it following nonsurgical embryo transfer in buffalo. Donor buffalo were superovulated with FSH, and embryos collected nonsurgically were evaluated for stage of development and quality. They were transferred nonsurgically to 91 recipients on Days 5 to 7 of the natural (n = 52) or induced (n = 39) estrus (estrus = Day 0). The overall pregnancy rate of 24/91(26.4%) was higher than in earlier reports for buffalo but was much lower than in cattle. Pregnancy rates were not affected by season (autumn vs winter), side of transfer (right vs left uterine horn), or type of estrus (spontaneous vs induced). The pregnancy rate was high 11/27(40.7%) when donors and recipients were closely synchronized, while it was compromised when recipients were in estrus at +12 h (1/7, 14.3%) and at -12 h (5/27, 18.5%). Asynchrony beyond 12 h on either side resulted into conception failure. The pregnancy rate tended to increase with the increase in CL size of recipients, while stage of embryonic development had no effect. The transfer of an 8-cell embryo with a 16-cell embryo led to the birth of heterosexual twins, indicating that the uterine milieu of Day 5 to 6 recipients may be tolerated by the out-of-phase 8-cell embryo, at least in the presence of a more mature embryo. Embryo quality had the greatest effect on pregnancy rate as it was higher (P < 0.005) after the transfer of Grade I than Grade III embryos (6/10, 60.0% vs 3/36, 13.9%). Assessment of returns to estrus indicated that among nonpregnant recipients, 17/67 (25.4%) embryos never matured sufficiently to prevent luteolysis through maternal recognition of pregnancy (MRP), while 14/67 (20.8%) embryos probably died following MRP. These results indicate that efforts to increase pregnancy rate following embryo transfer in buffalo should include prevention of luteolysis during the first week of transfer and a reduction in the incidence of embryonic mortality.     

Survival of 4-cell mouse embryos derived from in vitro fertilization after ultrarapid freezing and thawing

Four-cell mouse embryos obtained by fertilization in vitro were frozen ultrarapidly, immediately after one-step exposure to a highly concentrated solution (modified VS1) at room temperature, and later thawed in a 37 degrees C waterbath. The proportion of morphologically normal embryos on thawing was 95.7% (198/207), and 83.8% (166/198) of these embryos developed to morulas and early blastocysts after 24 hr in culture. All frozen-thawed embryos that developed to morulas and early blastocysts were transferred to the uterine horns of recipients on day 3 of pseudopregnancy and 47.0% (78/166) of transferred embryos developed to normal young.     

Cryopreservation of strains and mutant genes in mice

Three kinds of freezing methods were tested with embryos of DNI strain. The survival rate after thawing was 47.5%, 66.7% and 77.8% in the 2-step method, modified slow freezing method and modified 2-step method, respectively. Then, the modified 2-step method was applied to the embryos from 7 strains and a pair of interstrain crosses. PMSG treatment at the beginning of diestrus following HCG treatment after 48 hrs resulted in much yield of 8-16-cell embryos in all strains. The average number for each strain was as follows: DNI; 18.9, DDN; 13.0, BS; 20.4, C57BL/6; 12.9, DBA/2; 17.5, CRN; 19.8, PAN; 13.7 and DNI x C57BL/6-Ay; 21.7. Development of frozen-thawed embryos in culture varied among strains. Proportion of embryos that developed to the morula or blastocyst stage was as follows: DNI; 64.6%, DDN; 71.9%, BS; 53.6%, C57BL/6; 57.3%, DBA/2; 65.0%, CRN; 52.5%, PAN; 17.4% and DNI x C57BL/6-Ay; 44.1%. These results indicate that the ability of embryos to survive freezing and thawing is influenced by their genetic background. Live young were produced from DNI, DDN, BS and DNI x C57BL/6-Ay embryos after transfer to recipients. Comparative assessment of the developmental ability of frozen-thawed embryos after transfer among strains should be performed in further study.     

Laparoscopic transfer of ovine and cervine embryos using the transpic technique

A transpic technique was developed to transfer embryos to 352 sheep and 4 deer recipients using a laparoscope, a modified pair of Allis forceps and a modified Cassou aspic normally used for laparoscopic uterine insemination. The overall proportion of uncomplicated transfers in Experiment 1 in 216 recipient ewes was 90.7% (range between groups 80 to 100%), 3.7% of the transfers were presumed to be loss of embryos during expulsion from the transpic, and 5.6% were apparent transfers into the uterine wall. In Experiment 2,83% of transfers into 136 ewe recipients were uncomplicated, 5% were presumed to be loss of embryos during expulsion, 1% was apparent transfer into the uterine wall, and 11% involved 2 attempts at transfer. Only 34% of 116 recipients receiving low-quality frozen-thawed embryos were pregnant and 24% of the 226 embryos survived to term. In contrast, high pregnancy rates (>80%) and embryo survival rates (>70%) were achieved following uncomplicated and twice attempted transfers of fresh embryos. Pregnancy rates and embryo survival rates were low (<2%) following the presumed loss of embryos during expulsion and apparent transfers into the uterine wall. All 4 deer transfers were uncomplicated and 2 2 good-quality embryos survived to term compared with 0 2 low-medium quality embryos. The transpic technique is a moderately invasive technique which permits fast (15 to 20/h) and reliable transfer of embryos in small ruminants. With appropriate care, nearly all of the embryos can be correctly placed in the uterus, and high pregnancy rates and embryo survival rates can be achieved using this technique.     

Two-step freezing of two-cell rabbit embryos after partial dehydration at room temperature

The effect of rapid freezing and thawing on the survival of 2-cell rabbit embryos was examined. When embryos in 2.2 M-propanediol were directly plunged from room temperature to liquid nitrogen some of them survived after thawing (8%) but only if they had been pretreated by exposure to an impermeable solute, sucrose, that makes the blastomeres shrink osmotically before cooling. High survival (77-88%) in vitro was obtained when pretreated embryos were first held at -30 degrees C for 30-240 min before immersion into liquid nitrogen. Transfer of such frozen-thawed embryos gave a survival rate to live young similar to that obtained with controls (26% and 32% respectively). DMSO was less effective than propanediol; only 2 out of 38 sucrose-pretreated frozen-thawed embryos developed in vitro. The present work shows that a combination of partial dehydration of blastomeres at room temperature with their permeation by a cryoprotective agent offers a simple method for successful rapid freezing and thawing of rabbit embryos.     

Plasma progesterone profiles and factors affecting embryo-fetal mortality following embryo transfer in dairy cattle

The relationship between plasma progesterone (P4) levels and embryo survival, and the value of P4 profiles for the selection of cattle embryo transfer recipients is still a matter of controversy. This study reports a comparison between lactating cows and heifers (n = 407) from a single dairy herd, after transfer of either fresh or frozen-thawed good quality embryos, of their ability to sustain embryo-fetal development to term. Plasma P4 concentrations on the day of estrus (Day 0 = D0), Day 4, Day 7 and on Day 21 were measured and related to embryo survival. Plasma P4 levels on Days 0, 4 and 7 were similar in recipients later found pregnant or open. Plasma P4 levels on Day 7 were significantly higher (P < 0.01) in heifers than in cows, but they were similar in pregnant and nonpregnant heifers and in pregnant and nonpregnant cows. Pregnancy rates for fresh and frozen-thawed embryos were higher in heifers than in cows, but the differences did not reach significance. However, the overall late embryonic mortality was significantly higher (P < 0.01) and the calving rate for frozen-thawed embryos was significantly lower (P < 0.05) in cows than in heifers. As expected, plasma P4 on Day 21 was significantly higher (P < 0.001) in pregnant than in nonpregnant recipients, but there was no difference between pregnant cows and pregnant heifers. Plasma P4 levels on Day 7 of recipients presumed pregnant on Day 21 and later found pregnant or nonpregnant were similar, but plasma P4 levels on Day 21 were significantly higher (P < 0.001) in pregnant than in nonpregnant recipients. The results of this study suggest that plasma P4 levels until the day of transfer, except for the rejection of recipients with abnormal luteal function, are of limited practical use for embryo transfer recipient selection. However, in lactating cows low plasma P4 values on Day 7 might negatively affect embryo survival, while in heifers this effect is not noticeable. Lactating cows are more prone to embryo loss than heifers, especially in the case of frozen-thawed embryos; this is associated with a lower competence of the corpus luteum at Day 7.