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The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40 V/mm, 20 μs each) followed by 6‐dimethylaminopurine (6‐DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION) + 6‐DMAP + CHX (12.07%) activation was higher than that of ION + 6‐DMAP (8.6%) activation or ION + CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION + 6‐DMAP + CHX and DC + 6‐DMAP + CHX groups. The blastocyst rate of ION + 6‐DMAP + CHX‐activated oocytes in the basic rabbit culture medium (M‐199) + 10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M‐199 + 10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3–5 days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6–9 days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.  相似文献   

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To study the effects of equine chorionic gonadotropin (eCG) on pubertal development, 38 Milchschaf spring born male lambs fathered by the same ram and grazing over native pasture as a single mob during all the experiment were divided into three groups as follows: group 100 (100 IU of eCG weekly i.m., n = 11), group 400 (400 IU of eCG weekly i.m., n = 12) and group 0 (controls, n = 15). Lambs were weighed and scrotal circumference was registered every 2 weeks since birth until 170 days of age (end of experiment). On days 125 and 167 semen was collected using an electroejaculator. Semen volume and concentration, mass and individual sperm motility (scale 0 to 5) and total number of spermatozoa in the ejaculate were recorded. The sexual behaviour of the lambs was evaluated twice, on days 127 and 170 in a pen test with oestrous ewes. There were no significant effects of treatment on body weight or scrotal circumference, semen characteristics or sexual behaviour. At least in the administration regimens tested, eCG treatment has no effect on prepubertal reproductive development of male lambs.  相似文献   

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The aim of this study was to evaluate the reproductive performance of gilts that had a similar age but different weights at the onset of puberty stimulation by boar exposure at 144 days. Gilts were divided into two groups according to their lifetime growth rate from birth to approximately 144 days of age. Mean growth rates at this moment were 577 and 724 g/day for group 1 (G1; n = 58) and group 2 (G2; n = 58), respectively. After selection, gilts were weighed at approximately 155, 165 and 175 days of age, on the insemination day and at slaughter. Gilts were inseminated, on average, at 193 days of age and were slaughtered 32 days after insemination, when the number of corpora lutea and embryos were recorded. Higher growth rate gilts (G2) reached puberty earlier (155.3 vs 164.1 days; p < 0.01). More gilts of G2 group attained puberty by 190 days of age (p = 0.004) than G1 gilts (95%; 55/58 vs 76%; 44/58). The anoestrous rate, until 60 days after the onset of boar exposure was higher (p < 0.01) in G1 (19.0%; 11/58) than in G2 (3.4%; 2/58) group. However, there were no differences in the pregnancy rate (90.7 vs 94.5), ovulation rate (15.9 vs 16.5), total embryos (12.9 vs 11.7), viable embryos (12.0 vs 11.1) and embryo survival (73.7% vs 68.5%), between G1 gilts and G2 gilts, respectively (p > 0.05). High growth rate gilts attain puberty earlier and have a lower anoestrous rate than low growth rate gilts.  相似文献   

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The objectives of this study were to assess the efficiency of polarized light microscopy (PLM) in detecting microtubule‐polymerized protein in in vitro‐matured bovine oocytes; to examine its effects on oocyte developmental competence; and to assess the meiotic spindle of in vitro‐matured oocytes after vitrification/warming and further assessment of oocyte developmental competence. In the first experiment, the presence of microtubule‐polymerized protein (MPP) was confirmed as a positive PLM signal detected in 99.1% of analysed oocytes (n = 115), which strongly correlated (r = 1; p < 0.0001) with the presence of MPP as confirmed by immunostaining. In the second experiment, oocytes (n = 651) were exposed or not (controls) to PLM for 10 min and then fertilized and cultured in vitro. Oocytes exposed to PLM did not significantly differ from controls with regard to cleavage, total blastocyst and expanded blastocyst rates and cell numbers. In the third experiment, meiotic spindles were detected in 145 of 182 oocytes (79.6%) following vitrification and warming. Interestingly, after parthenogenetic activation and in vitro culture, oocytes that displayed a positive PLM signal PLM(+) differed significantly from PLM(?) in cleavage and Day 8 blastocyst rates. These results suggest that polarized light microscopy is an efficient system to detect microtubule‐polymerized protein in in vitro‐matured bovine oocytes and does not exert detrimental effects on bovine oocyte developmental competence. Moreover, PLM could be used as a tool to assess post‐warming viability in vitrified bovine oocytes.  相似文献   

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The de Wildt Cheetah and Wildlife Centre was established in 1971 and the first cheetah cubs were born in 1975. During the period 1975–2005, 242 litters were born with a total of 785 cubs. Mean cub survival from 1 to 12 months and greater than 12 months of age was 71.3 and 66.2%, respectively. The majority of losses (84.9%) occurred during the first month postpartum whereas only 15.1% deaths took place between 1 and 12 months of age. Females were first bred at an age of approximately 3 years, reached maximum reproductive age at 6–8 years, where after fertility declined. Males reached peak reproduction at 6 and maintained this for up to 12 years of age. Male fertility was best correlated with sperm morphology. During recent years, for practical purposes, males were allocated to 'good' (≥70% normal), 'fair' (40–70% normal) and 'poor' (<40% normal) categories according to sperm morphology count. The breeding males were selected from the good (preferably) and fair categories but poor category males were also used at times. Average litter sizes for 'good', 'fair' and 'poor' males were 3.44 (n = 21), 3.14 (n = 18) and 2.28 (n = 18), respectively. In females the heritability for litter size was high at 0.5848 (532 progeny, 1975–2007) and the maternal heritability for cub mortality was estimated to be 0.596. The data from the de Wildt Cheetah and Wildlife Centre and two other centres in the world (Kapama and Wassenaar) demonstrate that cheetah can be bred successfully in captivity.  相似文献   

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This study focused on the effects on subsequent reproductive traits of females and development of the current litter as a result of different contact between the rabbit doe and its progeny when a temporary doe–litter separation was used for biostimulation. Immediately after parturition and adjusting to eight young per litter, multiparous Pannon White rabbit does (n = 400) were divided into five groups based on parity, doe condition, and weight of litter and the kits. Rabbits in the control group (C) nursed freely up to weaning at 35 days of age. In the group following local farm practice (F) using a metal-plate for separation, females nursed once a day (8 am to 9 am) for the first 14 days of lactation and freely afterwards. In three biostimulation groups, there was a change from free to once a day nursing before insemination (i.e. controlled nursing at 8, 9 and 10 days) provided with a wire-mesh separation (BW: permits visual, olfactory, and acoustic contact); using a metal-plate for separation (BM: no visual but possible olfactory, acoustic and vibration contact); or with taking the litter with the nest-tray 5 m away from the doe's cage (BN: there is no contact at all) and returning to free nursing just after AI (at 11 days in the morning) up to weaning. The type of separation exerted a significant influence on oestrus and subsequent kindling of does. Comparing C, F, BW, BM and BN groups, sexual receptivity (30.4, 45.4, 24.0, 25.6, 43.0%, respectively; P < 0.05), fertility (74.4, 84.2, 80.8, 80.3, 89.5%; P < 0.05) and kindling rates (71.1, 85.5, 76.9, 77.3, 88.2%, respectively; P < 0.05) improved in F and BN groups. The total number of kits born (10.0, 10.3, 10.5, 10.4, 9.55, respectively; P = 0.392) and kits born alive per litter (9.25, 9.65, 9.59, 9.83, 9.03, respectively; P = 0.607) were not affected, but newborn kit weight was numerically less in the BW group (69.7, 67.5, 65.5, 67.6, 67.3 g, respectively; P = 0.166). Growth rates of current progeny up to weaning were higher and identical in C and BM but less in F and BN groups (27.5, 25.6, 27.1, 27.3, 26.7 g/day, respectively; P = 0.001). However, the total weight of 70-day-old rabbits per doe did not differ significantly (17.51, 17.53, 17.54, 16.81, 15.81 kg, respectively; P = 0.271). On the whole, the production of the F, BM and BN groups was superior to that of the control and BW groups since subsequent kindling results improved without a significant reduction in slaughter rabbits per doe from current litters.  相似文献   

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The aim of this study was to evaluate the effects of methionine supplementation on energy metabolism and reproductive performance during the early post‐partum period in primiparous does. Forty nulliparous New Zealand White does were used. Females were randomized in two groups at calving: the control group (n = 20) was fed with the basal diet, and the methionine group (n = 20) was fed the basal diet plus 1 g/animal/day of methionine from the day of calving to 4 days post‐partum. Results showed that methionine supplementation increased (p = 0.032) the concentration of insulin‐like growth factor‐1 with respect to control group 4 days post‐partum. It similarly increased the prolificacy (p = 0.03), the number of kits born alive per litter (p = 0.06) and the body gain weight of the litter during supplementation (p = 0.035). These results were observed despite the does in the methionine group having a deeper negative energy balance than the does in the control group. Finally, methionine supplementation did not affect receptivity (p = 0.23), fertility (p = 0.49), the number of kits born dead per litter (p = 0.86) insulin and metabolites as glucose, non‐esterified fatty acids and triglycerides. In conclusion, our results show that methionine supplementation during the first 4 days of the post‐partum period in rabbits increases total litter size and the corporal weight of kits and is associated with an increase in blood concentration of IGF‐1.  相似文献   

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Melatonin may play an important role in protecting gametes and embryos from the potential harmful effects of oxidative stress. In this study, we first examined two different heat stress (HS) treatments for in vitro oocyte maturation (Experiment 1: 38.5 vs 41.0°C, during the first 20 h; Experiment 2: 38.5 vs 41.5°C, during the entire period) on bovine oocyte maturation and embryo development. Second, we tested different melatonin concentrations added to the maturation and culture medium (Experiment 3: 0, 10?12, 10?9, 10?4 m ; Experiment 4: 0, 10?3 m ), both with and without HS (38.5 or 41.5°C, respectively). In Experiment 1, the HS treatment resulted in a lower maturation rate and number of cells/blastocyst (C/B) and a higher blastocyst rate than that in the control group. In Experiment 2, oocytes/embryos from heat‐stressed oocytes (HSO) had a lower maturation, cleavage and blastocyst rates, as well as a lower C/B compared with the control. In Experiment 3, in HSO groups, 10?4 m melatonin resulted in an increased blastocyst rate compared with 0 m melatonin, with a similar blastocyst rate to the non‐HSO without melatonin. Melatonin did not have any effect in embryos from non‐HSO groups compared with the control. In Experiment 4, 10?3 m melatonin produced lower cleavage and blastocyst rates in HSO and lower blastocyst rate in non‐HSO when compared to melatonin‐untreated oocytes/embryos. In conclusion, 10?4 m melatonin was found to alleviate bovine oocytes from the harmful effects of HS.  相似文献   

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Fifty‐five New Zealand white × California cross‐bred female rabbits were used to study the effect of protein level and flushing method on the reproductive performance of rabbits. The treatments consisted of three protein diets (18, 20 and 24% crude protein (CP)) and three flushing methods in a 3 × 3 factorial design. The flushing methods consisted of (i) flushing multiparous does and feeding a 16% CP diet during pregnancy; (ii) flushing multiparous does and feeding an 18% CP diet during pregnancy; and (iii) flushing nulliparous does and feeding an 18% CP diet during pregnancy. Flushing rabbits with different levels of protein did not significantly affect gestation length, litter size and mortality of kits. The trend showed an increase in litter weight with the increase in protein level from 18 to 24%. However, kits from does flushed with 24% CP had a higher individual kit weight gain than those on 20 and 18% CP. Gestation length, total litter size at birth, number of kits dead at birth and number of kits dead at 21 days post‐partum were not affected by flushing methods. There was, however, a significant effect of the flushing method on the number of kits alive at birth, and 7, 14 and 21 days post‐partum. Nulliparous does flushed and maintained on 18% CP during pregnancy had a significantly higher number of kits alive at birth, and 7, 14 and 21 days post‐partum. Litter weight was significantly higher for nulliparous does at 7, 14 and 21 days compared with that of multiparous does flushed and placed on a 16 or 18% CP diet during pregnancy. Protein intake during pregnancy had a significant effect on litter birthweight. Nulliparous and multiparous does flushed and placed on an 18% CP diet during pregnancy had a significantly higher litter birthweight than multiparous does flushed and placed on a 16% CP diet during pregnancy. The effect of protein level during pregnancy on the number of kits alive at birth was not significant. There was no interaction between protein level and flushing method on the reproductive performance of rabbits.  相似文献   

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The emergence of epizootic rabbit enteropathy is leading to changes in weaning protocols in commercial rabbitries. Traditional weaning protocols are being replaced with late weaning, beyond 35 days postpartum (dpp). The main objectives of this study were to compare the peripheral blood lymphocyte populations of multiparous rabbit does under two reproductive rhythms (insemination at 11 dpp and weaning at 28 dpp, insemination at 25 dpp and weaning at 42 dpp), and to assess the influence on those of kits. Samples of peripheral blood were taken in 22 adult females and 44 of their kits at different critical times, and several lymphocytic populations were evaluated by flow cytometry. Additionally, the perirenal fat thickness of does was also measured at partum and weaning to observe if body condition correlates with lymphocyte populations. During whole lactation, counts of total, CD5(+), CD4(+) and CD8(+) lymphocytes of females were generally lower with weaning at 42 dpp compared to 28 dpp. Moreover, counts of total, B and CD5(+) lymphocytes in rabbit does weaned at 42 dpp correlated to their body condition (+0.60 to 0.82; P<0.05), contrary to that observed in rabbit does weaned at 28 dpp. Some correlations between lymphocyte counts in both groups of does and weaning rabbits were observed. At weaning, those young rabbits weaned at 42 dpp had a significantly lower number of CD4(+) lymphocytes than those weaned at 28 dpp (P<0.01). In conclusion, the 42 ddp rabbit does presented a lower number of total lymphocytes and lymphocytic subpopulations during lactation and at weaning, as well as lesser capacity of adjustment during the gestation-lactation cycle.  相似文献   

14.
This investigation addresses the possibility of providing mouse embryos or other foreign objects with a protective mucin coat by transferring them into the oviduct of a life rabbit doe. Mouse embryos at the 8 or 16-cell stage, rabbit oocytes and latex spheres resembling mouse embryos in size were transferred to the ligated oviducts of ovulation-induced rabbit does. The does were killed 24 h later to have their oviducts flushed. A large proportion of the latex spheres (89%) and of the ovulated oocytes of the recipient does (92%) was recovered. The recovery rates for transferred rabbit oocytes, either intact or with the zona pellucida removed, were 61% and 51%, respectively, whereas that for mouse embryos was extremely poor (20%). Rabbit oocytes with or without zona were enveloped in a thick mucin coat regardless whether they had been transferred or ovulated by the recipients. The same applied to empty rabbit zonae. Mouse embryos and latex spheres were also covered by a mucin coat, but it was four times thinner. While residing in the rabbit oviduct, the mouse embryos continued developing to a stage comparable to what would have been expected in situ . During the subsequent in vitro culture, mouse embryos continued developing to the expanded blastocyst stage. They did, yet, not hatch from the zona. It may be concluded that particles of various origins, when placed into the oviduct of ovulated rabbit does, will be provided with a mucin covering which is, however, considerably thinner than that surrounding oocytes or zonae pellucidae originating from rabbits.  相似文献   

15.
In mid-September, 1 month before the insertion of intravaginal pessaries to induce sexual activity, blood samples were collected every 4 days from 16 ewe lambs aged 7 months, in order to determine the incidence of ovulations by measurement of plasma progesterone concentrations. It has been studied whether the response to a progestagen treatment of ewe lambs apparently close to puberty could be modified by the onset of the ovarian events preceding puberty. The effect of the presence or absence of ovulations prior to progestagen treatment on the potential reproductive performance (fertility, litter size and fecundity), embryo development [embryo quality and interferon-tau (IFNτ) secretion], luteal function (progesterone secretion in vitro ) and endometrial progesterone content was studied in seven ovulating (Ov+) and nine nonovulating ewe lambs (Ov−) on day 14 after mating. The best potential reproductive results were obtained with Ov+ animals, although these differences could not be initially attributed to either different progesterone secretion in vitro or concentration of endometrial progesterone. Irrespective of the experimental groups, secretion of progesterone by luteal tissue from ewe lambs with normal embryos was significantly greater (p<0.05) than that of animals with abnormal embryos or with no embryos. Normal embryos secreted a higher amount of IFN-τ than those embryos classified as abnormal (p<0.07). In conclusion, ewe lambs which exhibit luteal activity before puberty have the highest levels of reproductive performances after a progestagen treatment. Corpora lutea from ewe lambs with normal embryos had higher rates of progesterone secretion in vitro and their embryos had a higher IFN-τ production by the embryos, indicating greater capacity for subsequent development.  相似文献   

16.
Uterine capacity (UC), defined as the total number of kits from unilaterally ovariectomized does at birth, has a high genetic correlation with litter size. The aim of our research was to identify genomic regions associated with litter size traits through a genomewide association study using rabbits from a divergent selection experiment for UC. A high-density SNP array (200K) was used to genotype 181 does from a control population, high and low UC lines. Traits included total number born (TNB), number born alive (NBA), number born dead, ovulation rate (OR), implanted embryos (IE) and embryo, foetal and prenatal survivals at second parity. We implemented the Bayes B method and the associations were tested by Bayes factors and the percentage of genomic variance (GV) explained by windows. Different genomic regions associated with TNB, NBA, IE and OR were found. These regions explained 7.36%, 1.27%, 15.87% and 3.95% of GV, respectively. Two consecutive windows on chromosome 17 were associated with TNB, NBA and IE. This genomic region accounted for 6.32% of GV of TNB. In this region, we found the BMP4, PTDGR, PTGER2, STYX and CDKN3 candidate genes which presented functional annotations linked to some reproductive processes. Our findings suggest that a genomic region on chromosome 17 has an important effect on litter size traits. However, further analyses are needed to validate this region in other maternal rabbit lines.  相似文献   

17.
Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA‐1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA‐1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo‐derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA‐1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA‐1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8–16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA‐1A revealed that it shares 91–98% identity with other mammalian sequences. It can be concluded that higher level of HSPA‐1A mRNA in IVP embryos in comparison with in vivo‐derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA‐1A gene could be used as a stress biomarker during pre‐implantation development.  相似文献   

18.
Nowadays, the use of foetal calf serum (FCS) during in vitro embryo culture is very controversial. Whilst some authors have encouraged its use, others reject it because of its harmful effects. Although in vitro embryo production in red deer is a promising assisted reproductive technique, it is still in its infancy and a great effort is needed to update the protocols used. The aim of this study was to assess whether FCS supplementation in red deer embryo culture medium is necessary to produce blastocyst and, if so, when is the best time to add it in terms of blastocyst production and quality. In vitro blastocysts were cultured with FCS added at 24, 48 or 96 hours post‐insemination (hpi). In addition, a treatment without FCS was used as control. Six hundred and ninety‐four cumulus–oocyte complexes were collected for in vitro fertilization. Cleavage rate was examined at 48 hpi, and blastocyst yield was recorded on days 6, 7 and 8. FCS had no influence on cleavage and blastocyst rate for any of the treatments studied. However, the number of cells was higher (p = .025) in those blastocysts cultured with FCS from 48 hpi compared with FCS‐free culture media (93.88 ± 7.76 vs. 54.11 ± 8.36). In conclusion, the addition of FCS to the embryo culture medium at 48 hpi improves the quality of red deer blastocyst, although it does not affect the percentage of embryos obtained.  相似文献   

19.
This study assessed the effects of cryoprotectant concentration during equilibration on the efficiency of bovine blastocyst vitrification and the expression of selected developmentally important genes. In vitro produced bovine blastocysts were equilibrated in either 7.5% ethylene glycol (EG) + 7.5% DMSO (Va group) or in 2% EG + 2% DMSO (Vb group) then vitrified on Cryotop® sheets in 16.5% EG + 16.5% DMSO + 0.5M sucrose. After warming, embryos were cultured for 48 hr. Re‐expansion, hatching, and the numbers of total and membrane damaged cells were compared among vitrified groups and a control. There was no significant difference between the vitrified groups in survival, cell numbers and the extent of membrane damage. Vitrification increased the number of membrane‐damaged cells in both groups, however, in a greater extent in the Vb group. Vitrification increased (p < .05) the expression of the HSP70 gene in Va but not in Vb embryos. The expression of IGF2R, SNRPN, HDAC1, DNMT3B, BAX, OCT4, and IFN‐t genes were the same in control and vitrified groups. In conclusion, the concentration of cryoprotectants during equilibration did not affect survival rates; however, normal cell numbers could be maintained only by equilibration in 15% cryoprotectants which was associated with increased HSP70 expression.  相似文献   

20.
In a diabetic pregnancy, an altered maternal metabolism led to increased formation of reactive α‐dicarbonyls such as glyoxal (GO) and methylglyoxal (MGO) in the reproductive organs and embryos. The enzyme glyoxalase (GLO) 1 detoxifies reactive α‐dicarbonyls thus protecting cells against malfunction or modifications of proteins by advanced glycated end products (AGEs). The aim of this study was to analyse the influence of a maternal insulin‐dependent diabetes mellitus (IDD) on GLO1 expression and activity in preimplantation embryos in vivo and human trophoblast cells (Ac‐1M88) in vitro. Maternal diabetes was induced in female rabbits by alloxan before conception and maintained during the preimplantation period. GLO1 expression and activity were investigated in 6‐day‐old blastocysts from healthy and diabetic rabbits. Furthermore, blastocysts and human trophoblast cells were exposed in vitro to hyperglycaemia, GO and MGO and analysed for GLO1 expression and activity. During gastrulation, GLO1 was expressed in all compartments of the rabbit blastocyst. Maternal diabetes decreased embryonic GLO1 protein amount by approx. 30 per cent whereas the enzymatic activity remained unchanged, indicating that the specific GLO1 activity increases along with metabolic changes. In in vitro cultured embryos, neither hyperglycaemia nor MGO and GO had an effect on GLO1 protein amount. In human trophoblast cells, a stimulating effect on the GLO1 expression was shown in the highest GO concentration, only. Our data show that maternal diabetes mellitus affects the specific activity of GLO1, indicating that GLO1 was post‐translationally modified due to changes in metabolic processes in the preimplantation embryos.  相似文献   

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