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1.
The survival rate of vitrified germinal vesicle (GV) stage porcine oocytes is very low, and it is not known if the vitrification damages the nucleus, cytoplasm or both. We have evaluated the eventual GV or cytoplasmic damage in fully grown (FG) and growing vitrified oocytes. Fifty-five percent of nonvitrified FG cumulus-denuded oocytes reached the metaphase II (MII) stage in culture. When growing oocytes from preantral (PA) and early antral (EA) follicles were matured in vitro, almost all oocytes were arrested at the GV stage (GV stage: PA 88.9 and EA 79.5%, respectively). When fresh GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes and matured in vitro, some of them reached the MII stage (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). The maturation rate of vitrified FG oocytes was only 6.1% but increased dramatically when vitrified GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes (MII stage: VitFG/FG 43.9%, VitPA/FG 7.1% and VitEA/FG 26.3%, respectively). These results were not significantly different from those for the nonvitrified groups (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). We activated the reconstructed oocytes that received fresh or vitrified GVs (FG/FG, EA/FG, VitFG/FG and VitEA/FG) and examined their embryonic development. Cleaved embryos (nonvitrified groups 13.0-61.8%, vitrified groups 33.3-40.0%) and blastocysts (nonvitrified groups 0.0-18.2%, vitrified groups 0.0-2.9%) were obtained after activation. These results demonstrate that vitrified porcine GVs maintain maturational and developmental competence and that vitrification predominantly damages the cytoplasm.  相似文献   

2.
Joining immature gamete cryopreservation and germinal vesicle transplantation (GVT) technique could greatly improve assisted reproductive technologies in animal breeding and human medicine. The present work was aimed to assess the most suitable cryopreservation protocol between slow freezing and vitrification for immature denuded bovine oocytes, able to preserve both nuclear and cytoplasmic competence after thawing. In addition, the outcome of germinal vesicle transfer procedure and gamete reconstruction was tested on the most effective cryopreservation system. Oocytes, isolated from slaughterhouse ovaries, were stored after cumulus cells removal either by slow freezing or by vitrification in open pulled straws. After thawing, oocytes were matured for 24 h in co-culture with an equal number of just isolated intact cumulus enclosed oocytes, and fixed in order to evaluate the stage of meiotic progression and cytoskeleton organization. Our results showed that after warming, vitrified oocytes reached metaphase II (MII) in a percentage significantly higher than oocytes cryopreserved by slow freezing (76.2% and 36.5% respectively, p < 0.05). Moreover, vitrification process preserved the organization of cytoskeleton elements in a higher proportion of oocytes than slow freezing procedure. Therefore vitrification has been identified as the elective method for denuded immature oocytes banking and it has been applied in the second part of the study. Our results showed that 38.3% of oocytes reconstructed from vitrified gametes reached the MII of meiotic division, with efficiency not different from oocytes reconstructed with fresh gametes. We conclude that vitrification represents a suitable method of GV stage denuded oocyte banking since both nuclear and cytoplasmic components derived from cryopreserved immature oocytes can be utilized for GVT.  相似文献   

3.
The aim of this research was to identify the changes in the cytoplasmic ultrastructure of immature and matured oocytes in buffalo (Bubalus bubalis ). Oocytes were matured in vitro in tissue culture medium?199 with and without sericin, and then analyzed by light and transmission electron microscopy. The experiment result showed that the nuclear maturation rate of buffalo oocytes was significantly higher in the presence of sericin (80.6%) than without sericin (68.1%) (P  < 0.05). The immature oocytes were characterized by cortical granule clusters in the ooplasm and the absence of perivitelline space (PVS ). In contrast, the oocytes matured either with or without sericin showed the formation of PVS , erected microvilli, the migration of cortical granules to the cytoplasmic periphery, and the clear appearance of the mitochondria and vesicle in the oolemma. Interestingly, matured oocytes with sericin have smaller cortical granules than do immature oocytes (P  < 0.05). In conclusion, supplementation of 0.05% sericin in the maturation medium can enhance the maturation rate of buffalo oocytes. Several cytoplasmic ultrastructures were relocated and modulated during the in vitro maturation process of buffalo oocytes: PVS development, cortical granules migration to periphery, and mitochondria and vesicles in the cortical region. The ultrastructure was similar between the groups with and without sericin.  相似文献   

4.
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5.
In vivo matured oocytes collected by laparoscopic follicular aspiration (LFA) from hormone treated female goats were used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Japanese native (Shiba) goats were used as donor females and some donor females were used repeatedly (two or three times) at intervals of a few months. To induce synchronization of estrus, a sponge containing 0.5 g of progesterone was inserted into the vagina of each goat for 14 days. These animals were also treated with follicle stimulating hormone (FSH) in a series of 8 injections over 4 days. The first FSH injection was administered on the morning of day 9 of sponge insertion. On the morning of day 13, 50 µg of gonadotropin‐releasing hormone (GnRH) was injected into each animal. Twenty‐nine hours after GnRH injection, LFA was performed. After removal of cumulus cells, collected oocytes with the first polar body were selected and enucleated for nuclear transfer. Anterior pituitary cells isolated from an adult male Shiba goat were transfected with a DNA fragment containing the enhanced green flourescent protein gene and the puromycin resistance gene. A single donor cell was inserted into the perivitelline space of each enucleated oocyte and fusion was induced with one electric pulse of 20 V for 10 µs. The SCNT goat eggs were cultured in chemically defined medium at 38.5°C in 5% CO2, 5% O2, 90% N2 for 9 days. By LFA, 396 oocytes were collected from a total of 30 females. After removal of cumulus cells, 64% of them extruded the first polar body. The percentage of SCNT goat eggs produced using in vivo matured oocytes which developed to the blastocyst stage (20–21%) was significantly higher (P < 0.05) than that produced with in vitro matured oocytes (3–8%).  相似文献   

6.
Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.  相似文献   

7.
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   

8.
Lyophilization has been used since long time to preserve yeast and bacteria strains. Subsequently, a great deal of efforts has been dedicated to the preservation in a dry state of red blood cells and platelets. However, despite more than 30 years passed by, no significant progress has been achieved. Recently, it has been reported that freeze-dried mice spermatozoa were able to generate normal offspring following injection into the mature mice oocytes. In this work, we prompted to apply the lyophilization protocol developed for mice spermatozoa to sheep somatic cells (lymphocytes and granulosa cells). More than 350 enucleated sheep oocytes were injected with granulosa cells, and freeze dried using the protocol developed for mice sperm cells. Transplanted nuclei organized large pronuclei with fragmented DNA, but none of them entered the first mitosis. In the second part of the experiments, trehalose and EGTA were found to reduce significantly the extent of nuclear damage (65% and 55% intact nuclei in lymphocyte and granulosa cells, respectively) following freeze drying. Granulosa cells lyophilized with EGTA/trehalose and stored at room temperature for 3 years were used for nuclear transfer, and the injected oocytes were cultured in vitro for 7 days. Approximately 16% of the oocyte injected with freeze-dried cells developed into blastocysts. To conclude, we demonstrated for the first time that nucleated cells maintain genomic integrity after prolonged storage in a dry state, and we were able to achieve early embryonic development following injection of these cells into enucleated sheep oocytes.  相似文献   

9.
Inhibitors of cyclin‐dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus‐oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor‐free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (?) at 38.5°C and 5% CO2. At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/?) exhibited total expansion while in both Rosco groups (+/?) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/?). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.  相似文献   

10.
探讨程序化冷冻与玻璃化冷冻对小鼠GV期卵母细胞及二细胞期胚胎的复苏率及其发育潜能的影响。通过小鼠的卵母细胞与早期胚胎的不同冷冻方法的比较,为后续阿旺绵羊的胚胎冷冻保存提供参考。采用程序化冷冻与玻璃化冷冻技术,分别冷冻小鼠GV期卵母细胞及二细胞期胚胎,复苏后培养,比较不同冷冻处理后的复苏率、成熟率与囊胚率。小鼠GV期卵母细胞程序化冷冻复苏率(48.00%±5.29%)显著低于玻璃化冷冻复苏率(65.00%±5.00%),有统计学差异(P=0.0147<0.05);而程序化冷冻后复苏卵母细胞的发育成熟率略高于玻璃化冷冻组,但无统计学意义。小鼠二细胞期胚胎程序化冷冻组复苏率(76.00%±2.00%)显著高于玻璃化冷冻组复苏率(70.00%±2.00%),有统计学差异(P=0.0213<0.05);冷冻后复苏胚胎发育的囊胚率程序化冷冻略低于玻璃化冷冻及对照组,但无统计学意义。  相似文献   

11.
We investigated the effects of different selective α2‐adrenergic receptor (AR ) agonists (detomidine, medetomidine, xylazine, and brimonidine) on the contractions of horse‐isolated bronchi induced by electrical field stimulation (EFS ) and by carbachol. No effects were observed on the contraction induced by carbachol, while α2‐AR agonists reduced EFS ‐evoked contractions in a concentration‐related fashion. The rank order of potency (pD 2) was brimonidine (7.40 ± 0.20) >medetomidine (7.09 ± 0.24) >detomidine (6.13 ± 0.55) >xylazine (4.59 ± 0.16). The maximal effects (Emax) were ?56.3% ± 6.3%, ?40.4% ± 6.9%, ?48.6% ± 9.9%, and ?72.7% ± 12.7% for brimonidine, medetomidine, detomidine, and xylazine, respectively. Adrenergic block by guanethidine enhanced the potency (8.10 ± 0.05, 7.30 ± 0.15, 6.83 ± 0.41, and 5.40 ± 0.22) and the efficacy (?95.2% ± 0.7%, ?45.2% ± 11.7%, ?58.5% ± 9.8%, and ?97.9% ± 0.6%) of brimonidine, medetomidine, detomidine, and xylazine, respectively. Selective α2‐AR antagonist, atipamezole, competitively antagonized the inhibition of EFS ‐evoked contractions induced by all agonists except xylazine. These results suggest the existence of presynaptic α2‐AR s on cholinergic neurons, negatively regulating the release of acetylcholine in horse bronchial muscle, and that α2‐AR agonists may be beneficial against vagally mediated bronchoconstriction.  相似文献   

12.
The objective of this study was to evaluate the effect of two sources of commercial porcine pituitary‐derived follicle‐stimulating hormone (pFSH) and pFSH—porcine Luteinizing Hormone (pLH), including equine chorionic gonadotropin (eCG), in ovulatory and embryonic response in Pelibuey sheep. Twenty‐four Pelibuey sheep were used and were assigned randomly to four treatments (n = 6): (T1; 200 mg pFSH‐Folltropin®); (T2; 200 mg pFSH + 300 UI eCG‐Folligon®); (T3; 250 UI pFSH/pLH‐Pluset®) and (T4; 250 UI pFSH/pLH + 300 UI eCG). The interval of hours from withdrawal of the device to the beginning of oestrus (BO) was lower (p < .05) in sheep treated with eCG (T2 = 8.0 ± 1.4 and T4 = 10.0 ± 2.8) than in those without eCG (T1 = 12.6 ± 0.6 and T3 = 20.6 ± 2.4). The ovulatory rate (OR) was higher (p < .05) in T1 = 15.5 ± 2.8 and T2 = 15.6 ± 1.4, compared to T3 = 8.1 ± 3.2 and T4 = 11.8 ± 2.8; a significant difference was not shown between them (T1 vs. T2 and T3 vs. T4) when including eCG. The number of non‐fertilized oocytes (NFO) was lower (p ? .05) in T1 = 0.8 ± 0.4 and T3 = 1.8 ± 1.8, compared to those that included eCG (T2 = 6.3 ± 2.4 and T4 = 2.1 ± 1.2). The number of transferable embryos (TE) was higher (p < .05) when FSH was applied (T1 = 5.8 ± 1.1), compared with (T2 = 2.6 ± 1.1, T3 = 2.3 ± 1.4 and T4 = 2.8 ± 1.5). The commercial treatments (pFSH or pFSH‐pLH) in combination with eCG did not improve OR, NFO and TE. However, the exclusive pFSH (Folltropin) treatment presented a higher OR, lower number of NFO and higher number of TE.  相似文献   

13.
Studies were conducted to examine the possibility of preserving slaughterhouse‐derived buffalo ovaries at 4°C for 0 (control), 12 and 24 h to maintain the developmental competence of the oocytes (experiment 1), to assess the effect of incubation temperature during oocyte maturation on rates of in vitro maturation (IVM) and in vitro fertilization (IVF) of buffalo oocytes and embryo development (experiment 2), and to examine the effect of storage at 25°C for 0 (control), 4 and 8 h of frozen–thawed buffalo sperm and BO and H‐TALP as sperm processing and fertilization media on cleavage and embryo development in vitro of buffalo oocytes (experiment 3) in order to optimize the IVF technology in buffalo. Results suggested that storage of ovaries at 4°C for 12 or 24 h significantly (p < 0.05) reduced the developmental potential of oocytes. Incubation temperatures during the IVM influenced the fertilization rate but had no significant effect on maturation and subsequent embryo development. The incubation temperature of 38.5°C during IVM was found to be optimum for embryo production in vitro. Storage of frozen–thawed sperm at 25°C for 8 h significantly (p < 0.05) decreased its ability to cleave the oocytes. Sperm processed in BO medium had significantly (p < 0.05) higher ability to cleave the oocytes than the H‐TALP medium.  相似文献   

14.
The objective of the present study was to investigate the effect of testicular tissue lysate (TTL) on developmental competence of germinal vesicle (GV) stage porcine oocytes. Two types of TTL were prepared through repeated freeze–thaw in liquid nitrogen, one from whole testicular tissue (wTTL) and other from either of four different sections of testes, namely just beneath the tunica albuginea (TA), from the transitional area between the seminiferous cord/tubules and the mediastinum testis (TR) and from the intermediate area (parenchymal tissue origin) and CE (cauda epididymis origin). The whole or section‐wise TTL treatments were given for 44 hr during in vitro maturation (IVM). Oocyte maturation was done in either of the two media, namely defined (high‐performance basic medium for porcine oocyte maturation, commercially available) and serum containing (TCM199). After maturation, oocytes were co‐incubated with fresh spermatozoa for 6 hr and then transferred to embryo culture media. Treatment of GV stage oocytes with wTTL (1 mg/ml) increased the cleavage and morula percentage rate (69.23 ± 6.23 and 48.15 ± 6.77, respectively) than that of their control (58.33 ± 8.08 and 32.54 ± 5.53, respectively) in defined media, and in serum‐containing media, cleavage and morula percentage rate were almost equal in both treatment (54.56 ± 7.79 and 34.70 ± 6.78, respectively) and control (59.52 ± 8.21 and 38.52 ± 6.54, respectively). However, effect of wTTL was not significant. In case of section‐wise TTL supplements, TR section significantly (p < .01) improved cleavage and morula rate (58.43 ± 7.98 and 36.14 ± 6.89, respectively) followed by TA. In conclusion, present study indicates that IVM, in vitro fertilization and in vitro culture of embryo are improved in the presence of TTL, particularly its TR section. Further study is expected to reveal the principal components of TTL which may prove useful for IVM.  相似文献   

15.
Routinely, swim‐up method is used to separate high‐quality sperm; however, long processing time and close cell‐to‐cell contact during the centrifugation step are inevitable elements of oxidative stress to sperm. The objective was to evaluate Sephadex? and glass wool filtration to separate motile, intact and viable sperm for in vitro fertilization in buffalo. The cumulus–oocyte complexes (COC s) were collected from ovaries of slaughtered buffaloes by aspiration and matured for 24 hr in CO 2 incubator at 38.5°C and 5% CO 2. Matured COC s were rinsed twice in fertilization TALP and placed in the pre‐warmed fertilization medium without sperm. Cryopreserved buffalo semen was thawed at 37°C for 30 s and processed through Sephadex?, glass wool filtration and swim‐up (control). Total and motile sperm recovery rates were assessed, resuspended in fertilization TALP and incubated for 15–20 min in CO 2 incubator. Samples prepared by each method were divided into two aliquots: one aliquot was studied for sperm quality (progressive motility, membrane integrity, viability, liveability), while the other was subjected to co‐incubation with sets of 10–15 in vitro matured oocytes. Data on sperm quality were analysed by ANOVA , while in vitro fertilizing rates were compared by chi‐squared test using SPSS ‐20. Least significant difference (LSD ) test was used to compare treatment means. Glass wool filtration yielded higher total and motile sperm recovery rate, while Sephadex? filtration improved (p  <  .05) sperm quality (progressive motility, membrane integrity, viability, liveability). Sperm preparation through Sephadex filtration yielded higher in vitro fertilization rate in terms of cleavage rate compared to glass wool filtration and swim‐up (control). In conclusion, cryopreserved Nili‐Ravi buffalo sperm selected through Sephadex filtration showed improved quality and yielded better fertilization rates (cleavage rate) of in vitro matured/fertilized oocytes. Sephadex filtration could be a promising technique for use in in vitro fertilization in buffalo.  相似文献   

16.
Kaempferol (KAE) is a natural flavonoid present in different plant species and exhibits anti‐inflammatory, antioxidant, and anticancer therapeutic properties. In the present study, we investigated the influence and underlying mechanisms of KAE supplementation on porcine oocytes during in vitro aging. The results show that KAE treatment can alleviate the aging‐related reduction of developmental competence. We observed that the blastocyst production rate in aged oocytes treated with 0.1 μM KAE was significantly higher than in untreated aging oocytes (36.78 ± 0.86% vs. 27.55 ± 2.60%, respectively, p < .05). The KAE‐treated aging oocytes had significantly reduced levels of reactive oxygen species (p < .05). Furthermore, the mRNA levels of the embryonic pluripotency‐related genes Oct4, NANOG, and ITGA5 were significantly increased in blastocysts derived from KAE‐treated oocytes (p < .05). During excessive oocyte culture, KAE treatment maintained the mitochondrial membrane potential and reduced apoptosis; however, this was not observed in untreated aging oocytes. In conclusion, our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.  相似文献   

17.
Current in vitro embryo production protocols in the Iberian red deer (Cervus elaphus hispanicus) need to be optimized; oocyte harvesting in situ followed by overnight holding could reduce the human effort and shipping costs. In our work, post‐mortem ovaries were retrieved, and the oocytes were harvested and allocated to G1 group (good quality) or G2 + G3 group (low quality). The oocytes were separately subjected to immediate in vitro maturation (IVM) or held overnight in a holding medium composed of 40% of TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts and 20% fetal bovine serum (FBS), at room temperature (16 hr). In vitro maturation was carried out in a basal medium supplemented or not with 50 ng/ml of epidermal growth factor (EGF). Our data showed that addition of EGF to the maturation medium increases the percentage of G1 oocytes reaching metaphase II (3.9% vs. 50%, basal vs. EGF; p < .001) and decreased their degeneration rate (69.9% vs. 22.2%, basal vs. EGF; p < .01) when oocytes were immediately matured. Overnight holding increased the meiotic competence of G1 oocytes (37.5% matured in basal medium) and EGF increased prophase arrest in G2 + G3 oocytes (16.1% vs. 38.8% in germinal vesicle [GV] stage in basal medium vs. EGF added medium; p < .05). Our data demonstrate that oocyte holding can be used in Iberian red deer oocytes. Interestingly, EGF addition increases the oocytes' meiotic competence in immediately matured oocytes but not after oocyte holding depending upon initial oocyte quality.  相似文献   

18.
To effectively control bovine mastitis, tilmicosin (TIL)‐ and florfenicol (FF)‐loaded solid lipid nanoparticles (SLN) with hydrogenated castor oil (HCO) were prepared by a hot homogenization and ultrasonication method. In vitro antibacterial activity, properties, and pharmacokinetics of the TIL‐FF‐SLN were studied. The results demonstrated that TIL and FF had a synergistic or additive antibacterial activity against Streptococcus dysgalactiae, Streptococcus uberis, and Streptococcus agalactiae. The size, polydispersity index, and zeta potential of nanoparticles were 289.1 ± 13.7 nm, 0.31 ± 0.05, and ?26.7 ± 1.3 mV, respectively. The encapsulation efficiencies for TIL and FF were 62.3 ± 5.9% and 85.1 ± 5.2%, and the loading capacities for TIL and FF were 8.2 ± 0.6% and 3.3 ± 0.2%, respectively. The TIL‐FF‐SLN showed no irritation in the injection site and sustained release in vitro. After medication, TIL and FF could maintain about 0.1 μg/mL for 122 and 6 h. Compared to the control solution, the SLN increased the area under the concentration–time curve (AUC0‐t), elimination half‐life (T½ke), and mean residence time (MRT) of TIL by 33.09‐, 23.29‐, and 37.53‐fold, and 1.69‐, 5.00‐, and 3.83‐fold for FF, respectively. These results of this exploratory study suggest that the HCO‐SLN could be a useful system for the delivery of TIL and FF for bovine mastitis therapy.  相似文献   

19.
The present work was conducted to examine (1) the morphology of dromedary cumulus‐oocytes complexes (COCs), (2) to study the incidence of spontaneous development of oocytes in vivo and (3) to assess the ability of in vitro matured dromedary oocytes to chemical parthenogenetic activation compared with in vitro fertilized (IVF) oocytes. COCs were recovered from dromedary ovaries classified according to their morphology into six categories. Oocyte diameter was measured using eye piece micrometer. For chemical activation, COCs with at least three layers of cumulus‐cells were in vitro matured (IVM) in TCM 199 + 10 μg/ml FSH + 10 IU hCG/ml + 10% FCS + 50 μg/ml gentamycin. COCs were incubated for 40 h at 38.5°C under 5% CO2 in humidified air. After IVM, matured oocytes with first polar body (first Pb) were divided into two groups. Group 1: activated in 7% ethanol (E) for 5 min followed by culture in 2 mM 6‐dimethylaminopurin (6‐DMAP, E D, subgroup 1) or 10 μg/ml cycloheximide (CHX, E CHX, subgroup 2) for 3.5 h at 38.5°C under 5% CO2. In group 2, oocytes were activated using 50 μM Ca A23187 (Ca A) for 5 min followed by culture in 2 mM 6‐DMAP (Ca D, subgroup 3) or 10 μg/ml CHX(Ca CHX, subgroup 4) for 3.5 h at 38.5°C under 5% CO2. For control group, IVM oocytes were fertilized using frozen‐thawed camel spermatozoa separated by swim‐up method then suspended in Fert‐TALP medium supplemented with 6 mg/ml BSA (FAF) + 10 μg/ml heparin. In all groups, oocytes were in vitro cultured in SOFaa medium + 5% FCS and 5 μg/ml insulin + 50 μg/ml gentamycin. Cleavage rate and embryo development were checked on Days 2, 5 and 8. An average of 11.3 ± 0.3 COCs were recovered/dromedary ovary. Categories 1 and 2 represented 33.1% and 34.8%, respectively, and were significantly higher (p < 0.01) than the other categories (19.1, 9.2 and 2.6% for categories 3–5, respectively). Category 6 (embryo‐like structures) represented 1.2% of the recovered oocytes, staining of these embryo‐like structures with orcien dye indicated the presence of divided cells with condensed nuclei. Dromedary oocytes averaged 166.2 ± 2.6 μm in diameter with black cytoplasm. Chemical activation of IVM dromedary oocyte with first Pb in 7% ethanol or 50 μM Ca A followed by culture in 2 mM 6‐DMAP showed significantly higher (p < 0.01) cleavage and developmental rates to the morula stage than oocytes activated using 7% ethanol or 50 μM Ca A followed by 10 μg/ml CHX or in vitro fertilized control group. Higher (p < 0.01) proportion of oocytes sequentially cultured in 10 μg/ml CHX or that in vitro fertilized were arrested at the 2–4‐cell stage compared with that cultured in 6‐DMAP.  相似文献   

20.
The objective was to investigate the effects of reproductive seasonality on gamete quality in plains bison (Bison bison bison). Epididymal sperm (n = 61 per season), collected during the breeding season (July–September), had significantly higher post‐thaw total motility (36.76 ± 14.18 vs 31.24 ± 12.74%), and lower linearity (0.36 ± 0.06 vs 0.39 ± 0.04) and wobbliness (0.49 ± 0.04 vs 0.51 ± 0.03; mean ± SD) compared to non‐breeding season (January–March) samples. Representative samples (n = 4) from each season were used in heterologous IVF trials using cattle oocytes. Cleavage, morulae and blastocyst percentage were higher for breeding vs non‐breeding season sperm samples (81.88 ± 6.8 vs 49.94 ± 6.77; 41.89 ± 13.40 vs 27.08 ± 23.21; and 30.49 ± 17.87 vs 13.72 ± 18.98%, respectively). Plains bison ovaries collected during the breeding (n = 97 pairs) and non‐breeding (n = 100 pairs) seasons were classified as luteal or follicular. Oocytes recovered from these ovaries were classified into five grades based on morphology. There was no significant difference in the number of luteal ovaries or grades of oocytes recovered. Oocytes were matured, fertilized (with frozen sperm from three bison bulls) and cultured in vitro. Cleavage percentage was higher for oocytes collected during breeding vs non‐breeding season (83.72 ± 6.42 vs 73.98 ± 6.43), with no significant difference in subsequent development to blastocysts. In summary, epididymal sperm from non‐breeding season had decreased total motility and resulted in reduced embryo production in vitro. Oocytes collected during non‐breeding season had reduced ability to be matured, fertilized and/or undergo cleavage in vitro. Data suggested that season influenced gamete quality in plains bison.  相似文献   

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