Effects of feed restriction during pregnancy on maternal reproductive outcome,foetal hepatic IGF gene expression and offspring performance in the rabbit

Primiparous female rabbits have high nutritional requirements and, while it is recommended that they are subjected to an extensive reproductive rhythm, this could lead to overweight, affecting reproductive outcomes. We hypothesised that restricting food intake during the less energetic period of gestation could improve reproductive outcome without impairing offspring viability. This study compares two groups of primiparous rabbit does in an extensive reproductive programme, one in which feed was restricted from Day 0 to Day 21 of gestation (R021), and another in which does were fed ad libitum (control) throughout pregnancy. The mother and offspring variables compared were (1) mother reproductive outcomes at the time points pre-implantation (Day 3 postartificial insemination [AI]), preterm (Day 28 post-AI) and birth; and (2) the prenatal offspring characteristic IGF system gene expression in foetal liver, liver fibrosis and foetus sex ratio, and postnatal factor viability and growth at birth, and survival and growth until weaning. Feed restriction did not affect the conception rate, embryo survival, or the number of morulae and blastocysts recovered at Day 3 post-AI. Preterm placenta size and efficiency were similar in the two groups. However, both implantation rate (P < 0.001) and the number of foetuses (P = 0.05) were higher in the R021 mothers than controls, while there was no difference in foetal viability. Foetal size and weight, the weights of most organs, organ weight/BW ratios and sex ratio were unaffected by feed restriction; these variables were only affected by uterine position (P < 0.05). Conversely, in the R021 does, foetal liver IGBP1 and IGF2 gene expression were dysregulated despite no liver fibrosis and a normal liver structure. No effects of restricted feed intake were produced on maternal fertility, prolificacy, or offspring birth weight, but control females weaned more kits. Litter weight and mortality rate during the lactation period were also unaffected. In conclusion, pre-implantation events and foetal development were unaffected by feed restriction. While some genes of the foetal hepatic IGF system were dysregulated during pregnancy, liver morphology appeared normal, and the growth of foetuses and kits until weaning was unmodified. This strategy of feed restriction in extensive reproductive rhythms seems to have no significant adverse effects on dam reproductive outcome or offspring growth and viability until weaning.     

In vivo development of vitrified rabbit embryos: Effects on prenatal survival and placental development

The aim of this work is to study the effect of the vitrification procedure on prenatal survival and on placental development at the end of gestation in rabbits (Oryctolagus cuniculus). One hundred eighty-one females were slaughtered at 72 h of gestation. Morphologically normal embryos recovered at 72 h of gestation were kept at room temperature until transfer or vitrification. Vitrified embryos (320 embryos) were transferred into a total of 24 does and fresh embryos (712 embryos) were transferred into a total of 43 does. Females were induced to ovulate 72 h before transfer when fresh embryos were transferred and 60 to 63 h before transfer when vitrified embryos were transferred. Each recipient doe received eight embryos into the left oviduct and eight embryos into the right oviduct. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at Day 14 of gestation. Recipient females were slaughtered by stunning and exsanguination 25 d after the transfer, and fetuses were classified according to their status. Live fetuses and fetal and maternal placenta were weighed Pregnancy rate was defined as the total number of females having at least one live fetus at Day 28 of gestation divided by the total number of females. Prenatal survival was estimated as live fetuses at Day 28 of gestation divided by the number of transferred embryos. The pregnancy rate after transfer of vitrified embryos (92%) was similar to that achieved with fresh embryos (86%), but prenatal survival was lower for vitrified than for fresh embryos (53% vs. 34%). We did not find differences in embryo survival from 72 h to implantation. Transfer of vitrified embryos reduced fetal survival from implantation to Day 28 (57% vs. 82%). Differences in the number of live fetuses at Day 28 of gestation were mainly due to the higher fetal mortality observed soon after implantation in pregnancies resulting from the transfer of vitrified embryos. A higher percentage of decidual reactions and atrophic maternal placentas (27.5% vs. 8.3%) and also of atrophic fetal and maternal placentas (12.1% vs. 5.3%) were observed after transfer of vitrified embryos. Both treatments showed similar percentage of dead fetuses (3.3% vs. 4%). Maternal placenta of the fetuses from fresh embryos was 15% heavier than maternal placenta of fetuses from vitrified embryos.     

Stanniocalcin (STC) in the endometrial glands of the ovine uterus: regulation by progesterone and placental hormones

Stanniocalcin (STC) is a hormone in fish that regulates calcium levels. Mammals have two orthologs of STC with roles in calcium and phosphate metabolism and perhaps cell differentiation. In the kidney and gut, STC regulates calcium and phosphate homeostasis. In the mouse uterus, Stc1 increases in the mesometrial decidua during implantation. These studies determined the effects of pregnancy and related hormones on STC expression in the ovine uterus. In Days 10-16 cyclic and pregnant ewes, STC1 mRNA was not detected in the uterus. Intriguingly, STC1 mRNA appeared on Day 18 of pregnancy, specifically in the endometrial glands, increased from Day 18 to Day 80, and remained abundant to Day 120 of gestation. STC1 mRNA was not detected in the placenta, whereas STC2 mRNA was detected at low abundance in conceptus trophectoderm and endometrial glands during later pregnancy. Immunoreactive STC1 protein was detected predominantly in the endometrial glands after Day 16 of pregnancy and in areolae that transport uterine gland secretions across the placenta. In ovariectomized ewes, long-term progesterone therapy induced STC1 mRNA. Although interferon tau had no effect on endometrial STC1, intrauterine infusions of ovine placental lactogen (PL) increased endometrial gland STC1 mRNA abundance in progestinized ewes. These studies demonstrate that STC1 is induced by progesterone and increased by a placental hormone (PL) in endometrial glands of the ovine uterus during conceptus (embryo/fetus and extraembryonic membranes) implantation and placentation. Western blot analyses revealed the presence of a 25-kDa STC1 protein in the endometrium, uterine luminal fluid, and allantoic fluid. The data suggest that STC1 secreted by the endometrial glands is transported into the fetal circulation and allantoic fluid, where it is hypothesized to regulate growth and differentiation of the fetus and placenta, by placental areolae.     

An experimental research on cryopreserving rabbit trachea by vitrification

Vitrification is a promising alternative to tissue preservation, in which the tissue is permeated with cryoprotective agents (CPAs) in order to circumvent the hazardous effects associated with ice formation. In this study, we evaluate the effect of vitreous cryopreservation of rabbit trachea, by comparing vitrification procedure with conventional computer-programmed slow freezing approaches. Harvested rabbit trachea were tailored and divided into groups and cryopreserved by vitrification and programmed freezing, respectively. The morphology and ultrastructure of the thawed tracheal fragments including HE dyes, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) staining, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were studied to assess the integrity of the tracheal fragments. Morphological studies demonstrated that both cryopreservation procedure retained the integrity of trachea, both epithelial cells, cilia and cartilage cells were in good shape. Compared with slow freezing methods, vitrification was less detrimental to cartilage cells and had a higher survival rate of chondrocytes and coverage of epithelium and cilia. Therefore, vitrification procedure can be a more satisfactory method to preserve trachea and the survival of chondrocytes in situ in cartilage tissue is adequate and respiratory epithelium is soundly present.     

Nucleic acid content and growth of fetal brain, liver and heart during inanition in pigs

Previous investigations have indicated that gilts deprived of dietary intake for periods up to 40 days are capable of maintaining pregnancy and producing offspring of normal body weight. The present experiment was designed to study the effects of inanition during middle or late pregnancy on growth and on protein and nucleic acid content of porcine fetal brain, liver and heart. Gilts were subjected to prolonged inanition (40 days; 0 kcal/day; water only) during either the middle third (Days 30-70, n = 3) or last third (Days 70-110, n = 3) of pregnancy; controls received a full diet (7028 kcal/day) until Day 70 (n = 3) or 110 (n = 3) when all dams were hysterectomized. Inanition during middle or late pregnancy had no detrimental effect on fetal brain development. Brain weight, cell size (protein/DNA ratio) and cell number (total DNA) were similar in all fetuses at Day 70 or 110. RNA concentrations at Days 70 and 110, protein concentration at Day 70 and total protein at Day 110 were higher in fetuses from starved dams than in those from controls, indicating greater protein synthetic activity in fetal brains from nutrient-deprived dams. Prolonged inanition during midpregnancy had only a limited effect on fetal liver and heart. Only liver RNA concentration and content at Day 70 differed in fetuses from starved dams; however, 40 days inanition during late gestation had marked detrimental effects. Liver weight, cell size and cell number were reduced in inanition as compared with controls by Day 110.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes in the intraplacental distribution of placental lactogen and alkaline phosphatase in the rat

The junctional and labyrinth regions of the rat chorioallantoic placenta during the second half of gestation showed different patterns of development with regard to DNA, protein, placental lactogen and alkaline phosphatase content. DNA and protein measurements indicated that growth of the labyrinth region was more rapid and persisted for longer during gestation than did growth in the junctional zone. At midpregnancy the junctional zone was the main source of placental lactogen whereas by late pregnancy both regions contributed considerable amounts. On Day 20 of gestation the labyrinth region contained significantly more placental lactogen than did the junctional zone. Alkaline phosphatase activity was predominant in the labyrinth zone throughout the second half of gestation. The results indicate that the chorioallantoic placenta is composed of two functionally distinct regions.     

Synthesis of 'decidualization-associated protein' in tissues of the rat uterus and placenta during pregnancy.

The synthesis of a soluble protein (referred to as 'decidualization-associated protein', DAP), has been examined in uterine and placental tissues of rats during pregnancy by means of polyacrylamide gel electrophoretic analysis of [3H]leucine-labelled soluble proteins. No synthesis of the protein was detected in non-implantation regions of the uterus. In implantation site tissue, no synthesis was detected on Days 6 or 7 of pregnancy. Only slight synthesis was present in the endometrium on Day 8, but synthesis rose rapidly from Days 9 to 12 in both the endometrium and myometrium although differences in the rates of increase were observed. Synthesis fell from Day 12 to 14 in both tissues. Synthesis by the myometrium was entirely localized in the mesometrial region, which contains the metrial gland. After Day 12, when the endometrium is represented by the chorioallantoic placenta, synthesis was examined in the labyrinthine and the decidua basalis/basal zone placenta tissues. No synthesis of 'DAP' was detected in the labyrinthine placenta from Day 16 of pregnancy. Synthesis observed in the decidua basalis/basal zone placenta fell dramatically from Day 14 to 20. The pattern of synthesis of 'DAP' during pregnancy suggests a role in the establishment of the chorioallantoic placenta and metrial gland in the rat.     

Effect of in vitro heat shock upon the synthesis and secretion of prostaglandins and protein by uterine and placental tissues of the sheep

The objectives of this study were to determine the secretion patterns of prostaglandins (PG) and protein during mid- (Day 100) and late- (Day 140) pregnancy in the ewe and to ascertain whether that pattern is altered by in vitro heat shock. Explant cultures were prepared from intercaruncular endometrium, caruncular endometrium, fetal cotyledon and interplacentomal placenta. Cultures were incubated at 39 or 42 degrees C for 18 h in the presence of arachidonic acid or L-[4,5(3)H]leucine. There were no effects of day of gestation or consistent effects of temperature upon de novo synthesis of tissue and secretory protein. Elevated temperature generally depressed PGE(2) secretion by maternal tissues and PGF secretion by caruncular endometrium but had little effect on PGE(2) release by fetal tissues or on PGF release by intercaruncular endometrium or fetal tissues. Day of gestation by tissue type interactions were found for PGF and PGE(2) release. At Day 100, maternal tissues secreted more PGF and PGE(2) than fetal tissues; at Day 140, PG secretion from fetal tissues was greater than at Day 100, and fetal PGE(2) release exceeded release from maternal tissues. Tissue proteins resolved by SDS-PAGE revealed the appearance in heat-shocked tissue of 93 and 72 kDa heat-shock proteins. In conclusion, elevated temperature depressed PGE(2) release, particularly from maternal tissues. Changes in PGE(2) suggest that the increase in utero-placental PGE(2) with increasing gestational age is due to changes in secretion of the fetal placenta.     

Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution

We designed and conducted a field trial to obtain accurate pregnancy rates of Day 7 bovine embryos after vitrification in PB1 containing 6.5 M glycerol and 6% BSA (w/v) and one-step dilution in 1 M sucrose compared with controlled slow freezing in 1.5 M glycerol and three-step dilution. Embryos were collected from superovulated donor cows, and Grade 1 and 2 morulae and blastocysts were randomly assigned to each cryopreservation treatment group. Dutch farmers were solicited to participate in the field trial by an advertisement that offered cryopreserved embryos at subsidized cost. Within a period of 11 wk, one of six technicians visited 150 farms. Standard nonsurgical methods were used to transfer a total of 728 cryopreserved embryos. Recipient cows, mainly multiparous and of various breeds, the so-called "bottom-end" of the national herd, received embryos either 6, 7 or 8 d after standing estrus during natural estrous cycles. We compiled a database on 22 factors that may influence establishment of pregnancy in order to check randomization of each factor over cryopreservation treatment groups and embryo transfer technicians and to perform the statistical tests. Overall pregnancy rates were 44.5% (n = 393) for vitrified embryos and 45.1% (n = 335) for slowly frozen embryos. Pregnancy rates were not significantly different (ANOVA, P = 0.79 or Chi- square analysis, P = 0.88). The registered data confirm that all factors were randomly distributed over cryopreservation methods and technicians. Technician was not a significant source of variation in pregnancy rate (analysis of variance, P = 0.79). Although three technicians performed better with the one-step procedure and the other three performed better using the three-step method, the interaction between the technician and cryopreservation method was not significant (Tukey's test for nonadditivity, P = 0.13). Our results indicate that 1) vitrification and one-step dilution can be successfully used in the field without significant reduction in the pregnancy rate and 2) the pregnancy rate obtained using the "bottom-end" of the herd is satisfactory for practical application.     

Foetal age determination and development in elephants

Elephants have the longest pregnancy of all mammals, with an average gestation of around 660 days, so their embryonic and foetal development have always been of special interest. Hitherto, it has only been possible to estimate foetal ages from theoretical calculations based on foetal mass. The recent development of sophisticated ultrasound procedures for elephants has now made it possible to monitor the growth and development of foetuses of known gestational age conceived in captivity from natural matings or artificial insemination. We have studied the early stages of pregnancy in 10 captive Asian and 9 African elephants by transrectal ultrasound. Measurements of foetal crown-rump lengths have provided the first accurate growth curves, which differ significantly from the previous theoretical estimates based on the cube root of foetal mass. We have used these to age 22 African elephant foetuses collected during culling operations. Pregnancy can be first recognized ultrasonographically by day 50, the presumptive yolk sac by about day 75 and the zonary placenta by about day 85. The trunk is first recognizable by days 85-90 and is distinct by day 104, while the first heartbeats are evident from around day 80. By combining ultrasonography and morphology, we have been able to produce the first reliable criteria for estimating gestational age and ontological development of Asian and African elephant foetuses during the first third of gestation.     

Chorionic expression of heterogeneous products of the PAG (Pregnancy-Associated Glycoprotein) gene family secreted in vitro throughout embryonic and foetal development in the pig

Porcine PAG (pPAG) are placental products of a multigene family that is strongly expressed in the chorionic epithelium (trophoblast and trophectoderm). The objective of this study was to define a pattern of the pPAG proteins, secreted in vitro by chorionic explants harvested on 16-77 days of pregnancy. Trophoblastic and trophectodermal explants were collected from pregnant (PR) gilts (n = 27) and used for protein in vitro production (8-261 h). Endometrial explants of luteal-phase gilts (E10, n = 4) and pseudopregnant gilts (PsE, n = 2) were used as negative controls for protein immunoblotting. Proteins (PR, E10, PsE) were isolated mainly from incubation media, fractionated, dialysed and separated by SDS-PAGE. Heterogeneous Western blotting with various polyclonal anti-PAG sera raised against bovine or ovine antigens (anti-bPAG, or anti-oPAG) initially identified the pPAG proteins. Such blotting of fractionated chorionic proteins allowed for the isolation of porcine antigens that were employed as immunogens to raise several homologous antisera (anti-pPAG). Crude antisera were adsorbed on endometrial extracts or proteins of non-PR pigs, to remove non-relevant antibodies. The patterns of pPAG proteins secreted in vitro varied throughout pregnancy (35-72 kDa). During implantation, approximately 43 kDa (Day 16) or approximately 68.1 kDa (Days 17-25) pPAG proteins were detected. During placentation and as pregnancy advanced (Days 31-77), approximately 72.3 kDa pPAG proteins were observed. The secretions of parallel multiple smaller proteins (35.4-47.2 kDa), presumably, as forms of processed pPAG precursors, increased with the progress of gestation. In conclusion, the pPAG protein family plays a very important role during implantation, placenta formation and embryonic/foetal development in the pig.     

Regulation of placental villous angiopoietin-1 and -2 expression by estrogen during baboon pregnancy

We recently showed an increase in vascular endothelial growth factor (VEGF), decrease in angiopoietin-1 (Ang-1) and unaltered Ang-2 expression by the villous placenta with advancing baboon pregnancy. Moreover, placental VEGF expression was increased by estrogen in early pregnancy. In the present study, we determined whether placental Ang-1 and Ang-2 are regulated by estrogen. Ang-1 and Ang-2 mRNA and protein were determined by RT-PCR and immunocytochemistry in the placenta of baboons on Day 60 of gestation (term is 184 days) after administration of estrogen precursor androstenedione on Days 25-59 or on Day 54 after acute estradiol administration. Chronic androstenedione treatment increased serum estradiol levels three-fold (P < 0.001) and decreased (P < 0.05) villous cytotrophoblast Ang-1 mRNA to a level (0.36 +/- 0.08 relative to 18S rRNA) that was one-third of that in untreated animals (0.98 +/- 0.26). Within 2 hr of estradiol administration, cytotrophoblast Ang-1 mRNA was decreased to a level (0.24 +/- 0.05) one-fifth (P < 0.05) of that in untreated animals (1.14 +/- 0.23). However, Ang-2 mRNA levels were unaltered. Ang-1, Ang-2 and estrogen receptors alpha and beta protein were localized within villous cytotrophoblasts providing a mechanism for estrogen action at this site. In summary, estrogen increased VEGF, decreased Ang-1, and had no effect on Ang-2 expression within placental cytotrophoblasts during early baboon pregnancy. We propose that the estrogen-dependent differential regulation of these angioregulatory factors underpins the unique pattern of neovascularization established within the villous placenta during primate pregnancy.     

LC‐MS/MS‐based serum proteomics for identification of candidate biomarkers for hepatocellular carcinoma

Associating changes in protein levels with the onset of cancer has been widely investigated to identify clinically relevant diagnostic biomarkers. In the present study, we analyzed sera from 205 patients recruited in the United States and Egypt for biomarker discovery using label‐free proteomic analysis by LC‐MS/MS. We performed untargeted proteomic analysis of sera to identify candidate proteins with statistically significant differences between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. We further evaluated the significance of 101 proteins in sera from the same 205 patients through targeted quantitation by MRM on a triple quadrupole mass spectrometer. This led to the identification of 21 candidate protein biomarkers that were significantly altered in both the United States and Egyptian cohorts. Among the 21 candidates, ten were previously reported as HCC‐associated proteins (eight exhibiting consistent trends with our observation), whereas 11 are new candidates discovered by this study. Pathway analysis based on the significant proteins reveals upregulation of the complement and coagulation cascades pathway and downregulation of the antigen processing and presentation pathway in HCC cases versus patients with liver cirrhosis. The results of this study demonstrate the power of combining untargeted and targeted quantitation methods for a comprehensive serum proteomic analysis, to evaluate changes in protein levels and discover novel diagnostic biomarkers. All MS data have been deposited in the ProteomeXchange with identifier PXD001171 ( http://proteomecentral.proteomexchange.org/dataset/PXD001171 ).     

Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification

We cryopreserved infantile rat ovaries by vitrification and assessed their viability by autotransplantation. Hemilateral ovarian transplantation was performed on rats on postnatal Days 10 to 12. The left ovary of each rat was dissected out, cryopreserved by vitrification using a modified vitrification solution (VS1), and then autotransplanted under the capsule of the right kidney. The right ovary of each rat was removed. For the control, the left ovary was dissected out from each rat and was immediately transplanted by the same procedure, without cryopreservation. Rats were nursed until weaning, and then the day of vaginal opening, estrous cyclicity from the day of vaginal opening until postnatal Day 84, and histology of ovarian grafts at postnatal Day 84 were examined. The time course of development of endocrine function of cryopreserved grafts was similar to that of fresh grafts. In ovarian transplants recovered on postnatal Day 84, antral follicles and corpora lutea (CL) were observed in addition to small follicles, although the number of antral follicles in cryopreserved grafts was smaller than in the fresh grafts. These results indicate that cryopreservation of ovarian tissue by vitrification can be used for the preservation of fertility and endocrine function of ovaries.     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型,研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化.基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征——DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14、0.49和1.43,与妊娠早期相比,妊娠中、晚期胎盘基因组DNA断裂值有显著性增加.TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层.免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax∶Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势.实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax∶Bcl-2的比例相关.     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型。研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化,基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征-DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14,0.49和1.43,与妊娠早期相比,妊娠中,晚期胎盘基因组DNA断裂值有显著性增加,TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层,免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax:Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势,实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax:Bcl-2的比例相关。     

Conventional slow freezing, vitrification and open pulled straw (OPS) vitrification of rabbit embryos

Three different methods of cryopreservation viz., conventional slow freezing, vitrification and open pulled straw vitrification were compared for their ability to support post thaw in vitro and in vivo development of rabbit embryos. Morula stage rabbit embryos were collected from super-ovulated donor does. They were randomly allocated to different freezing methods and stored up to 3 months in liquid nitrogen. After thawing and removal of cryoprotectants, embryos exhibiting intact zona pellucida and uniform blastomeres were considered suitable for in vitro culture and/or transfer. Three to five cryopreserved embryos placed in approximately 1 ml of culture medium (TCM 199 supplemented with foetal calf serum and antibiotics) were incubated for up to 72 h under humidified atmosphere of 5% CO2 in air at 39 degrees C. Development to hatched blastocyst stage was considered the initial indicator of success of cryopreservation of embryos. Of the embryos cryopreserved by programmed freezing, open pulled straw vitrification, vitrification-55 h pc and vitrification-72 h pc 55, 71, 17 and 48%, respectively, developed into hatched blastocysts. Similarly 19, 29, and 4% of embryos cryopreserved by programmed freezing, open pulled straw vitrification and vitrification -72 h pc developed into live offspring on transfer to recipient does. This is the first report on open pulled straw vitrification of rabbit embryos. Present results, suggest that (a) open pulled straw vitrification supports better in vitro survival of frozen thawed rabbit morulae; (b) both programmed freezing and OPS are similar but superior to vitirification in supporting in vivo survival of frozen thawed rabbit embryos.