牛体外受精胚的玻璃化保存及细胞遗传学研究

用25％的乙二醇和0.2mol/L蔗糖与25％忆二醇混合的玻璃化溶液对牛体外受精的早期囊胚用1步法冷漠保存，融解后，在D－PBS液（含有0.5mol蔗糖）中15min除去冷冻保护剂，培养48和96h。结果表明：用含有0.2mol的蔗糖和25％的乙二混合的玻璃化溶液保存的早期囊胚的生态力明显高于不含蔗糖的玻璃化溶液（P＜0.001）。染色体分析结果表明：含有蔗糖的玻璃化溶液保存的囊胚的卵裂细胞数明显高于单独使用25％乙醇保存的囊胚（P＜0.05）。染色体异常发生率无明显变化。     

Vitrification with Glutamine Improves Maturation Rate of Vitrified / Warmed Immature Bovine Oocytes

The current study examined the protective effects of l ‐glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 μg/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. l ‐glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).     

Ultra‐Structural Alterations in In Vitro Produced Four‐Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification

Cryopreservation is the process of freezing and preserving cells and tissues at low temperatures. Controlled slow freezing and vitrification have successfully been used for cryopreservation of mammalian embryos. We investigated the effect of these two cryopreservation methods on in vitro produced four‐cell stage bovine embryos which were classified according to their quality and separated into three groups. The first group was maintained as untreated controls (n = 350). Embryos of the second (n = 385) and the third (n = 385) groups were cryopreserved either by controlled slow freezing or by vitrification. Embryos in groups 2 and 3 were thawed after 1 day. Hundred embryos were randomly selected from the control group, and 100 morphologically intact embryos from the second and third group were thawed after 1 day and cultured to observe the development up to the blastocyst stage. The blastocyst development rate was 22% in the control group, 1% in the slow‐freezing group and 3% in the vitrification group. Remaining embryos of all three groups were examined by light microscopy, transmission electron microscopy and immunofluorescence confocal microscopy with subsequent histological staining procedures. Cryopreservation caused degenerative changes at the ultra‐structural level. Compared with vitrification, slow freezing caused an increased mitochondrial degeneration, cytoplasmic vacuolization, disruption of the nuclear and plasma membrane integrity, organelle disintegration, cytoskeletal damage, a reduced thickness of the zona pellucida and a formation of fractures in the zona pellucida. Further studies are required to understand and decrease the harmful effects of cryopreservation.     

小鼠胚胎玻璃化冷冻保存及保存时间对其体内外发育率的影响

本试验采用乙二醇为主体的ＥＦＳ４０玻璃化溶液,对小鼠扩张囊胚实行二步法玻璃化冷冻保存。结果保存１小时、１年和２年的胚胎,在体内外的发育率无显著性差异（Ｐ＞０．０５）。与对照组相比较也无显著性差异（Ｐ＞０．０５）。     

Effects of Developmental Stage, Embryonic Interferon-τ Secretion and Recipient Synchrony on Pregnancy Rate after Transfer of in vitro Produced Bovine Blastocysts

Three separate trials of bovine embryo transfers were performed consisting of 32, 41 and 33 transfers, respectively, to examine the effects of (a) the developmental stage of in vitro‐derived blastocysts, (b) the amount of interferon‐τ (IFN‐τ) they secreted during culture and (c) the cyclic stage of the recipient at the time of transfer on the probability of establishment of pregnancy. One blastocyst was transferred into the ipsilateral uterine horn to the CL. At the time of transfer, blastocysts were classified into one of three developmental stages (early blastocyst, blastocyst and expanded blastocyst) and the cyclic stage of each cow was assessed (?12 h, on time, +12 h, +24 h, >24 h). Prior to the second and third trials, blastocysts were individually cultured for 24 h in 50 μl medium droplets and the IFN‐τ concentration in the droplet was determined. Logistic regression analyses revealed that expanded blastocysts had a significantly higher likelihood of establishing pregnancy (p = 0.009), and that there was a significant interaction with the cyclic stage of the recipient in this group with lower rates of pregnancy resulting from decreasing synchrony with the recipient (p = 0.033). IFN‐τ secretion during culture was significantly higher in expanded blastocysts than in the other two groups (p < 0.05). A significant effect of the pre‐transfer level of IFN‐τ secretion was found only in the ‘Blastocyst’ group where transfer of embryos with lower IFN‐τ production prior to transfer resulted in higher pregnancy rates (p = 0.047). These results demonstrate that IFN‐τ secretion may be a useful tool to predict pregnancy outcome, but only within certain developmental stages.     

绵羊体内外受精胚胎玻璃化冷冻保存

用 EFS4 0溶液对绵羊体内外受精的早期囊胚进行了玻璃化冷冻保存的研究。经卵母细胞体外成熟、体外受精和体外培养获得的早期囊胚 ,分别作如下处理 :( A) 1 0 %EG中预处理 5min后 ,在 EFS4 0平衡 3 0 s(两步法 ) ;( B) EFS4 0中平衡 1 min(一步法 ) ;( C) EFS4 0中平衡 2 min(一步法 )。然后投入液氮中冷冻保存 ,解冻胚胎的继续发育率分别为 80 %、78%和 50 %,A、B2组的结果与对照组 ( 88%)无显著性差异 ( P >0 .0 5)。经超数排卵获得的体内受精早期囊胚用两步法冷冻保存 ,解冻后胚胎发育继续率为 89%,与对照组( 93 %)也无显著性差异 ( P >0 .0 5)     

Arachidic Acid in Extender Improves Post‐thaw Parameters of Cryopreserved Nili‐Ravi Buffalo Bull Semen

Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post‐thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post‐thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili‐Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris–citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 10⁶ spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5‐ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose‐dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome‐intact live sperm (%) and sperm chromatin integrity (%) were better (p < 0.05) in extender having 5.0 ng/ml of arachidic acid compared with control. At 10.0 ng/ml, these values did not vary (p > 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome‐intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post‐thaw quality parameters of cryopreserved Nili‐Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post‐thaw semen quality parameters was observed at 20.0 ng/ml.     

Solid‐Surface Vitrification and In‐Straw Dilution After Warming of In Vitro‐Produced Bovine Embryos

Three experiments were designed to test a solid‐surface vitrification system for bovine in vitro‐produced embryos and to develop a simple method of in‐straw dilution after warming, which can be potentially used for direct transfer in the field. Experiment 1 evaluated embryo survival rates (i.e. re‐expansion and hatching) after vitrification and warming in three different solutions: VS1 (20% ethylene glycol (EG) + 20% propanediol (PROH) + 0.25 m trehalose (Tr)), VS2 (20% EG + 1M Tr) or VS3 (30% EG + 0.75 m Tr). Re‐expansion and hatching rates were higher (p < 0.05) for embryos vitrified in VS3 (72.2 ± 1.9 and 58.2 ± 0.8) than VS1 (64.4 ± 0.9 and 37.2 ± 2.5) or VS2 (68.5 ± 1.5 and 49.6 ± 1.0; p < 0.05). Experiment 2 was designed to compare two methods of vitrification: glass micropipettes or solid surface, using the VS1 or VS3 solutions. No significant differences were detected between the two methods; but re‐expansion and hatching rates were higher (p < 0.05) with VS3 (73.5 ± 3.1 and 47.1 ± 2.1) than VS1 (63.3 ± 3.3 and 39.7 ± 2.8). In experiment 3, embryos were vitrified by solid surface in VS1 or VS3 solutions and cryoprotectants were diluted in‐straw after warming in a TCM 199, 0.25 m sucrose solution or holding media. Survival rates of embryos vitrified in VS3 did not differ between those exposed to 0.25 m sucrose (74.7 ± 1.3 and 57.2 ± 2.2) or holding (77.3 ± 1.4 and 58.0 ± 2.5) medium after warming; however, survival rates of embryos vitrified in VS1 were higher (p < 0.05) in those exposed to 0.25 m sucrose (67.7 ± 2.3 and 47.0 ± 1.7) than holding medium (54.5 ± 1.0 and 27.7 ± 3.1). In conclusion, solid‐surface vitrification using simplified EG‐based solutions and in‐straw dilution with holding media may be a practical alternative for cryopreservation and direct transfer of in vitro‐produced bovine embryos.     

Nitric Oxide and Superoxide Anion Production During Heparin‐Induced Capacitation in Cryopreserved Bovine Spermatozoa

The aim of this work was to quantify NO, O₂^? and ONOO^? production during heparin‐induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin‐dependent capacitation, O₂ uptake, and NO production. Conversely, O₂^? production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O₂ consumption (5.9 ± 0.6 nmol/min × 10⁷ cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 10⁷ cells), and a five‐fold increase in O₂^? production (1.3 ± 0.07 nmol/min × 10⁷ cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O₂^? generation and the second‐order rate constant of the reaction between these species. To conclude, heparin‐induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O₂ uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O₂^? production by a membrane‐bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation.     

Supplementation of Slow‐Release Melatonin Improves Recovery of Ovarian Cyclicity and Conception in Summer Anoestrous Buffaloes (Bubalus bubalis)

The role of melatonin as a protective neurohormone against restoring cyclicity in summer anoestrous animals in photoperiod species has gained wider acceptance. This study was designed to uncover the evidence the slow‐release melatonin (MLT) has on initiation of ovarian cyclicity and conception rate (CR) in summer anoestrous buffaloes. Thus, buffaloes diagnosed as summer anoestrous (absence of overt signs of oestrus, concurrent rectal examination and radioimmunoassay for serum progesterone at 10 days interval) were grouped as untreated (Group I, sterilized corn oil, n = 8) and treated (Group II, single subcutaneous injection of MLT @18 mg/50 kg bwt in sterilized corn oil, n = 20). Animals treated and detected in oestrus were artificially inseminated (AI) followed by division into Group III (second dose of MLT on 5th day post‐AI, n = 8) and Group IV (no melatonin administration, n = 10). Blood samples were collected at 4 days interval for estimation of serum MLT, progesterone and oestrogen using radioimmunoassay kit. Mean oestrous induction rate (OIR), oestrous induction interval (OII), interoestrous interval (IOI) and CR were estimated. Compared to control, concentration of melatonin was significantly (p < 0.05) higher in treated group ranging from 14.34 ± 1.72 to 412.31 ± 14.47 pg/ml whereas other two hormones did not show any concentration difference. Melatonin‐administered buffaloes showed significantly (p < 0.05) higher (90%) OIR with OII of 18.06 ± 1.57 days. Results showed improvement in conception rate in buffaloes administered with post‐insemination melatonin. It can be concluded from the study that slow‐release melatonin supplementation restored cyclicity in summer anoestrous animals resulting in improvement in conception rate in buffaloes.     

Early Cleaved Bovine Embryos Show Reduced Incidence of Chromosomal Aberrations and Higher Developmental Potential on Day 4.5 Post‐Insemination

The present study aimed to investigate whether the timing of the first zygotic cleavage (FZC) influences the speed of embryo development expressed by the total cell count and the rate of chromosomally aberrant embryos. Bovine embryos were produced in vitro and divided into two categories according to the timing of FZC: early cleavers (at 30 hpi; EC) and non‐early cleavers (at 48 hpi; NEC). On day 4.5 pi, embryos were grouped into three classes depending on the number of blastomeres: delayed (<8 BL), normal (8–16 BL) and advanced (>16 BL). We applied fluorescence in situ hybridization (FISH) with probes for bovine chromosomes 6 and X. The only form of chromosomal imbalance observed was mixoploidy [(2n/3n; 2n/4n); 19.9%, 54/271]. Early cleavers were less often chromosomally unbalanced (13.9%, 20/144) than their NEC counterparts (26.7%, 34/127). Among embryos developing at a normal speed, the NEC embryos were more often abnormal (NEC 20/80; EC 10/79; p < 0.05). The advanced embryos were not observed among the NEC category, whereas such embryos from EC category displayed no chromosomal aberrations. The majority of embryos arrested at the 8 BL stage were of NEC category and were carriers of chromosomally abnormal blastomeres. With regard to embryonic sex, we demonstrated that although males dominate among bovine embryos developing in vitro, the incidence of mixoploidy was equal for both sexes. It can be suggested that a good‐quality bovine embryo is usually an early cleaver that develops at higher speed and contains less aberrant cells. The present study also confirmed the usefulness of the FZC as a marker of embryo quality by demonstrating a significantly lower incidence of aberrations in early embryos.     

Evidence of Damage in Cryopreserved and Fresh Bovine Embryos Using the Tunel Technique

The objective of the present study was to evaluate the quality of bovine embryos cryopreserved in different years in Chiapas, Mexico. The embryos were obtained from a government institution (FIMEGEN) dedicated to promoting embryo transfer among dual-purpose cattle farmers. Forty-three embryos frozen in 1988, 1989, 2000 and 2002 were analysed with the Tunel technique to detect programmed cell death (apoptosis). Eleven fresh embryos were used as controls. Analysis of variance was used in embryos stored in the different years with averages tested using Tukey's test. Student's t-test was employed to compare fresh and frozen cells. Embryos with shorter storage time presented a lower number (p < 0.001) of Tunel-positive cells compared with embryos stored for longer time. On the contrary, when comparing the number of apoptotic cells between frozen and fresh embryos a higher number of positive cells (p < 0.05) were found in the former. The present results suggest that the cryopreservation per se caused damage that compromises the viability of the embryo. Another explanation for the lower pregnancy rate found in the tropics could be irreversible damage caused by poor storage technique in these large operations.     

Effects of Serum-Free In Vitro Maturation of Bovine Oocytes on Subsequent Embryo Development and Cell Allocation in Two Developmental Stages of Day 7 Blastocysts

Maturation of oocytes and the subsequent outcome of the in vitro production (IVP) are affected by the composition of in vitro maturation (IVM) medium. To determine the use of serum interfering with effects of single molecules, we aimed at developing simplified IVM medium. The experimental IVM media were: (1) M199-medium supplemented with hormones and serum (control), (2) as 1 but serum was substituted with fatty acid-free serum albumin (FAFBSA) and (3) M199-medium without hormonal and serum supplementation (M199). The quality of embryos was assessed on day 7 by morphology and cryotolerance, as well as by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) and differential staining. Results showed that the nuclear maturation was suppressed in M199 group alone. Embryo cleavage and development rates, and the proportion of quality 1 blastocysts were lower in the FAFBSA and M199 groups compared to the control. Differences in the cell allocation of fresh embryos were observed at the blastocyst stage, but not at the expanded blastocyst stage. The control group blastocysts had larger number of cells allocated to the inner cell mass (ICM), and the FAFBSA group blastocysts larger apoptotic cell proportion compared to the blastocysts derived from other groups. After cryopreservation, the reduction of ICM proportion and increase of apoptotic cell proportion of embryos were equal between the experimental groups. In conclusion, exclusion of serum from the IVM media reduces embryo development and may cause perturbations in blastocyst development. Differences in the cell allocation of blastocysts between IVM media may appear only when the developmental stages are taken into account.     

Methyl‐β‐Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida‐Binding Ability of Frozen/Thawed Bovine Spermatozoa

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein‐free media. However, the additive capacitating effect of methyl‐β‐cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen–thawed bovine spermatozoa in a BSA‐containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose‐dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre‐incubation of spermatozoa in MβCD‐supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)‐binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen–thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.     

Antibody‐Dependent Killing of Virus‐Infected Targets by NK‐Like Cells in Bovine Blood

Natural killer (NK) cell lysis of target cells by an Fc receptor‐mediated mechanism has not been conclusively demonstrated in cattle (Campos and Rossi, Vet. Immunol. Immunopathol. 8, 351–362, 1985), although it is well recognized in other species (Sulica et al., Nat. Immun. 14, 123–133, 1995). To resolve this problem, bovine peripheral blood mononuclear cells were completely depleted of adherent monocyte/macrophage type cells. The resulting enriched population of lymphocytes, was totally devoid of adherent monocytes, but contained up to 2 % NK‐like cells. On their own, this population had very low background levels of cytotoxicity for virus‐infected target cells in ⁵¹chromium release assays, but following the addition of virus‐specific antibodies, high levels of lysis were observed. This enhanced level of antibody‐dependent cytotoxicity demonstrated that bovine NK‐like cells can mediate killing of targets by an Fc receptor‐mediated mechanism as has been demonstrated for NK cells from other species.     

Effect of mSOF and G1.1/G2.2 Media on the Developmental Competence of SCNT‐Derived Bovine Embryos

The objective of this study was to compare the effect of two culture media: modified synthetic oviductal fluid (mSOF) and G1.2/G2.2, on the developmental competence of bovine somatic cell–cloned embryos. Cloned embryos were produced by transferring adult skin fibroblasts into enucleated MII oocytes. After activation, the reconstructed embryos were randomly allotted to either mSOF or G1.2/G2.2 for culture (the embryos were transferred from G1.2 to G2.2 on days 3 of culture). The development competence of cloned embryos in these two culture systems was compared in terms of cleavage rate, blastocyst formation rate and apoptosis cell number in day 7 blastocyts. To investigate the in vivo developmental competence of cloned embryos in the two culture systems, a total of 87 and 104 blastocysts derived from mSOF and G1.2/G2.2 medium groups were transferred individually to recipient Angus cows, respectively. No differences were observed in terms of cleavage rate, day 7 blastocyst rate and blastocyst cell number between these two culture systems. However, the day 6 blastocyst formation rate was significantly higher in G1.2/G2.2 than that in mSOF. In addition, blastocysts cultured in mSOF have a higher percentage of apoptotic blastomeres compared to those in G1.2/G2.2 (8.5 ± 1.2 vs 16.8 ± 1.5, p < 0.05). Although difference in pregnancy rate was not observed 40 days after embryo transfer, significantly higher pregnancy rate was observed in G1.2/G2.2 group after 90 days of embryo transfer (12.4% vs 37.5%, p < 0.05). Moreover, calving rate was significantly improved in G1.2/G2.2 group compared to mSOF group (27.9% vs 6.7%, p < 0.05). In conclusion, our results indicate that G1.2/G2.2 can improve developmental competence of bovine SCNT embryos both in vitro and in vivo, which is more suitable for culture of bovine SCNT embryos than mSOF medium.