The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.     

Survival of small and large littermate blastocysts in swine after synchronous and asynchronous transfer procedures

Thirty-two crossbred sows were assigned to synchronous and asynchronous embryo transfer procedures to determine if, within a litter, small blastocysts were as viable as large blastocysts. Synchronous embryo transfers were established when donors and recipients displayed the onset of estrus (Day 0) within 6 h of each other. Asynchronous transfers were established when recipients displayed the onset of estrus 18 to 24 h after that of donors. An equal number (four or five) of the smallest and largest diameter blastocysts, from a Day 7 donor, were transferred to separate uterine horns of a Day 7 (synchronous) or a Day 6 (asynchronous) recipient. Each recipient's uterine horns were ligated at the external bifurcation to prevent transuterine embryonic migration. The percentage of blastocysts surviving was determined 300 h (12.5 d) after donors exhibited estrus. Small as well as large Day 7 blastocysts survived following asynchronos transfer to a Day 6 recipient. However, fewer (P<0.01) small blastocysts survived synchronous transfer than large blastocysts. These data suggested that small blastocysts were lost due to asynchrony with the uterine environment; however, when transferred to a less advanced environment, small blastocysts were equally viable as large blastocysts.     

Reduced survival in utero from transferred mouse blastocysts compared with morulae

Studies on the survival of mouse embryos revealed that fewer offspring were produced when blastocysts, rather than morulae, were transferred to foster mothers. Approximately 8–10 h after fertilization F1 hybrid eggs (C57BL/6J × LT/Sv) were collected and cultured to morulae (day 4) or blastocysts (day 5 ) before transfer into uteri of day 3 foster mothers. A few recipients were killed on day 8 of gestation and deciduae were examined histologically. Embryos developing from transferred morulae were found to lie deep within the deciduae and were surrounded by numerous, large blood islands. Conversely, embryos developing from transferred blastocysts implanted more distally to the maternal blood vessels with only a few blood islands surrounding the embryos. These observations, suggesting abnormal implantation with insufficient embyro nourishment, were confirmed when uteri of foster mothers were examined on day 19 of gestation. Although the proportion of implantations from transferred morulae or blastocysts was similar (42 % and 47%, respectively), significantly more of the implantations were resorbed after transfer of blastocysts (78%) as compared with morulae (15%). These results demonstrate that transfer of day 5 cultured blastocysts into uteri of foster mothers increases embryonic mortality as a consequence of improper implantation.     

Sex-dependent loss of bisected bovine morulae after culture and freezing.

The objective of this study was to determine the post-transfer survival rate of bovine embryos cultured between the time of bisection and freezing. In this experiment 158 morulae were bisected and both portions were cultured for 24-44 h either in vivo after transfer to sheep oviducts (n = 80 morulae) or in vitro (n = 78 morulae) in Ham's F10 medium with 20% fetal calf serum, with bovine oviduct cells or in medium collected from oviduct cultures (conditioned medium). After culture, half of each morula was fixed for cytogenetic sex determination (n = 125) and the other half was frozen. The frozen halves were later thawed and transferred (n = 115) to recipients, who were, if pregnant, slaughtered to determine the sex of the fetus. The culture resulted in better pregnancy rates than those previously reported for embryos frozen immediately after bisection. The sex of 49 (33 males, 16 females) of the fixed demi-morulae was determined, and 38 of the transferred demi-morulae established pregnancies (23 males, 10 females and 5 fetuses that were not recovered). The male:female ratio in in vivo and in vitro culture groups was significantly different from the expected ratio of 1:1 and suggests that manipulation and culture of embryos results in a preferential loss of female embryos.     

Effect of addition of hyaluronan to embryo culture medium on survival of bovine embryos in vitro following vitrification and establishment of pregnancy after transfer to recipients

Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1 mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1 mg/mL, hyaluronan increased (P < 0.05) the percentage of oocytes that were blastocysts and re-expansion rate at 24 h after warming. At 0.5 mg/mL, hyaluronan tended (P < 0.10) to increase re-expansion rate at 48 h after warming and increased (P < 0.05) embryo hatching rate at 24 and 72 h. Treatment with BSA caused a slight reduction in cleavage rate (P < 0.05), but only for cultures containing hyaluronan (BSA × hyaluronan, P = 0.10), an increase in the percentage of oocytes that became blastocysts (P < 0.001), and a reduction in re-expansion rates (P < 0.001) and hatching rates (P < 0.05 or P < 0.01) at all times examined. In Experiment 2, embryos were produced in vitro and cultured in modified SOF containing 4 mg/mL BSA, with or without 1 mg/mL hyaluronan. At 159-162 h after insemination, grade 1 morula, blastocysts and expanded blastocysts were harvested for embryo transfer. Harvested embryos were transferred individually to lactating Holstein recipients with a palpable corpus luteum on Day 7 after presumptive ovulation. There was an interaction (P < 0.05) between hyaluronan and embryo stage on pregnancy rate. Recipients that received morula and blastocyst stage embryos treated with hyaluronan had a higher pregnancy rate than recipients that received control embryos of the same stage. There was no effect of hyaluronan on pregnancy rates of recipients that received expanded blastocysts. In conclusion, addition of hyaluronan to embryo culture enhanced blastocyst yield, improved survival following vitrification, and enhanced the post-transfer survival of fresh morula and blastocyst stage embryos.     

Embryos produced from fertilization with bovine viral diarrhea virus (BVDV)-infected semen and the risk of disease transmission to embryo transfer (ET) recipients and offspring

Bovine diarrhea virus (BVDV) causes a variety of economically important enteric and infertility problems in cattle. For that reason, several countries have eradicated the disease, and some others have schemes in progress to achieve freedom. Although there is a considerable amount of information about the risk of BVDV transmission through contaminated semen used for artificial insemination (AI), there is no evidence to indicate whether the resulting embryos, when used for embryo transfer, can lead to the transmission of BVDV to recipients or offspring. For this experiment, semen from a bull persistently infected with BVDV (10⁵ 50% tissue culture infective doses/mL NY strain) was used for insemination (two times at estrus) of BVDV-seronegative, superovulated cows (N = 35). Embryos were collected 7 days after insemination and subsequently were washed according to the International Embryo Transfer Society recommendations or left unwashed. Out of 302 collected oocytes and embryos, 173 (57%) were fertilized and the remaining 129 (43%) had degenerated. Infectious BVDV was detected in 24% (17/71) of unwashed and 10% (8/77) of washed embryos, and in all (N = 11) follicular fluid samples, oviductal epithelial cells, endometrium, and corpora lutea tissues as determined by the virus isolation test. After transfer of 39 washed embryos to 27 BVDV-seronegative recipients, 12 (44%) cows became pregnant and 17 calves free of BVDV and BVDV antibodies, including five sets of twins, were born. After embryo transfer, all pregnant and nonpregnant recipients remained free of BVDV and antibodies. In conclusion, results herein suggest that BVDV can be transmitted by AI resulting in the production of some proportion of contaminated embryos. However, it appears that such embryos, when washed according to International Embryo Transfer Society and the World Organization for Animal Health guidelines do not cause BVDV transmission to recipients or their offspring.     

Effects of interval between fusion and activation, cytochalasin B treatment, and number of transferred embryos, on cloning efficiency in goats

To improve the efficiency of somatic cell nuclear transfer (SCNT) in goats, we evaluated the effects of the interval between fusion and activation (1 to 5 h), cytochalasin B (CB) treatment after electrofusion, and the number of transferred embryos on the in vivo and in vitro development of cloned caprine embryos. The majority of the reconstructed embryos had condensed chromosomes and metaphase-like chromosomes at 2 and 3 h after fusion; cleavage and blastocyst rates from those two groups were higher (P < 0.05) than those of embryos activated 1, 4, or 5 h after fusion. Treatment with CB between fusion and activation improved in vitro and in vivo development of nuclear transfer (NT) goat embryos by reducing the fragmentation rate (P < 0.05). Although there were no significant differences in NT efficiency, pregnancy rate and kids born per recipient were increased by transfer of 20 or 30 embryos per recipient compared with 10 embryos. We concluded that CB treatment for 2 to 3 h between fusion and activation was an efficient method for generating cloned goats by somatic cell NT. In addition, increasing the number of embryos transferred to each recipient resulted in more live offspring from fewer recipients.     

Effect of equilibration period on the viability of frozen-thawed mouse morulae after rapid freezing

Mouse morulae were frozen with 1.5-4.0 M glycerol + 0.25 M lactose solution by direct plunging into liquid nitrogen vapor 0.5-30 min after equilibration at room temperature. After thawing, embryos were cultured in vitro, and the highest survival rates were obtained after exposure for 3 min at 3.0 and 4.0 M and for 5 min at 1.5 and 2.0 M glycerol levels. Significant reductions in the survival rates (P less than 0.05) were observed when equilibration periods were extended for 3-5 min at 3.0 and 4.0 M and for 5-10 min at 1.5 and 2.0 M glycerol levels. These results clearly demonstrate that the equilibration time of embryos in glycerol-lactose mixture is one of the most important factors in the present rapid freezing conditions. To clarify the factors that lower embryo viability after prolonged equilibration, we performed further experiments on the effects of exposure to glycerol-lactose mixture on the developmental potential of embryos without freezing and on the volume changes of embryos during the exposure to glycerol solution with or without lactose. It was suggested that the detrimental effects of prolonged equilibration are due not only to the toxicity and osmotic injury of higher concentrations of cryoprotectant solution but also to the influx of water into embryonic cells caused by the hypotonic salt concentration of the extracellular (freezing) solution.     

How to build fast muscles: synchronous and asynchronous designs

In animals, muscles are the most common effectors that translateneuronal activity into behavior. Nowhere is behavior more restrictedby the limits of muscle performance than at the upper rangeof high-frequency movements. Here, we see new and multiple designsto cope with the demands for speed. Extremely rapid oscillationsin force are required to power cyclic activities such as flightin insects or to produce vibrations for sound. Such behaviorsare seen in a variety of invertebrates and vertebrates, andare powered by both synchronous and asynchronous muscles. Insynchronous muscles, each contraction/relaxation cycle is accompaniedby membrane depolarization and subsequent repolarization, releaseof activator calcium, attachment of cross-bridges and muscleshortening, then removal of activator calcium and cross-bridgedetachment. To enable all of these to occur at extremely highfrequencies a suite of modifications are required, includingprecise neural control, hypertrophy of the calcium handlingmachinery, innovative mechanisms to bind calcium, and molecularmodification of the cross-bridges and regulatory proteins. Sideeffects are low force and power output and low efficiency, butthe benefit of direct, neural control is maintained. Asynchronousmuscles, in which there is not a 1:1 correspondence betweenneural activation and contraction, are a radically differentdesign. Rather than rapid calcium cycling, they rely on delayedactivation and deactivation, and the resonant characteristicsof the wings and exoskeleton to guide their extremely high-frequencycontractions. They thus avoid many of the modifications andattendant trade-offs mentioned above, are more powerful andmore efficient than high-frequency synchronous muscles, butare considerably more restricted in their application.     

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.     

Survival of equine embryos transferred to normal and subfertile mares

To test the hypothesis that an abnormal uterine environment was a cause of early embryonic loss in subfertile mares, morphologically normal embryos were transferred to normal mares (n = 20) and subfertile mares (n = 20), and embryo survival rates were compared. Embryos were recovered nonsurgically at Days 7 to 8 postovulation and transferred surgically to normal and subfertile mares that had ovulated on the same day or within 2 d after a donor. Survival of transferred embryos was monitored by ultrasonography of the recipient mare's uterus from Day 9 through Day 28 postovulation. There were no significant differences (P > 0.5) in the embryo survival rates at Day 12 (11 20 vs 9 20 ) or Day 28 (10 20 vs 8 20 ) for normal or subfertile mares, respectively. The uterine environment of subfertile mares was apparently adequate to support the development of transferred embryos from Days 7 or 8 through Day 28 postovulation.     

三种鹭异步孵化与雏鸟生长的比较研究

根据1996～1999年的野外工作和实验饲养,研究了池鹭、白鹭、夜鹭3种鹭的繁殖、雏鸟生长和异步孵化对雏鸟生长的影响.3种鹭于4月上、中旬迁到常山、余杭鹭保护区,9月下旬陆续迁离.孵卵期池鹭为23.0 d(n=26),白鹭23.9 d(n=32),夜鹭25.3 d(n=34);孵化率池鹭为76.32%,自鹭86.96%,夜鹭95.45%,池鹭繁殖力为3.21只,白鹭3.38只,夜鹭3.50只.雏鸟体重增长与成体体形大小呈负相关.幼雏体重增长与孵化顺序相关.在生长早期(≤8 d)全部雏鸟正常发育,之后差异显著.第1和第2孵化雏鸟生长曲线相似,但明显高于第4雏鸟,表明较早孵化者获得食物能力较强,而迟孵出雏鸟有食物不足现象.该3种鹭孵育幼雏能力的最适度估计为3只左右.     

Calmodulin, synchronous and asynchronous release of neurotransmitter

Evidence collected from studies on a wide range of secretory cells suggests that calmodulin may play an important role in stimulus-secretion coupling. Work on synaptosomes, central synaptic preparations and chromaffin cell preparations indicates that calmodulin probably also acts as the intracellular Ca2+-receptor for secretion in neuronal cells, Ca2+-binding resulting in activation of protein kinases and phosphorylation of certain secretory vesicle proteins. Studies on the effects of calmodulin-binding drugs at peripheral synapses have given surprising results, particularly the finding that evoked (synchronous) transmitter release is not suppressed by calmodulin inhibition, though asynchronous release can be markedly inhibited. It is suggested that the insensitivity of synchronous release to drug treatment is due to the fact that only vesicle-bound calmodulin is involved in this form of transmitter secretion. Asynchronous release, however, involves recruitment of cytosolic calmodulin and can therefore be inhibited by calmodulin-binding drugs.     

Impact of embryo technologies on secondary sex ratio in rabbit

Increasing evidence indicates that assisted reproductive technologies (ARTs) disturb skewed sex-ratio and induce sex-dimorphic postnatal effects. Undoubtedly, the combination of multiple ovulation and embryo transfer (MOET) together with the use of vitrification technique (MOVET) is currently being used in breeding programs. However, since the first case of sex skewing reported in 1991, the accumulative and long-term transmission of skewed sex-ratio to future generations has not been thoroughly evaluated. Here we test as MOVET program induce a skewed sex ratio, and we consider skewed sex ratio transmission to future generations. To this end, we first evaluated the F1 generation, demonstrating that a MOVET program causes a severe imbalance skewed secondary sex ratio (SSR) towards male by 12%. This imbalanced persist after a second MOVET program (F2 generation), with an accumulative skewed SSR towards male by 25%. Finally, using a crossbred generation derived from crossing F1 males derived from a MOVET program with naturally-conceived (NC) females, we show that the imbalance skewed SRR persist. Bodyweight comparison between MOVET animals and NC counterparts revealed significant changes at birth, weaning and adulthood. However, there was a significant interaction between F2 MOVET animals and sex, demonstrating an apparent accumulative sex-dimorphic effect. At adulthood, MOVET derived males presented a lower body weight. In conclusion, we show that the MOVET program causes a direct, accumulative and long-term transmission of skewed SSR.