Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa

The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4-6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution.     

Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro

The effects of media (TCM199 vs. synthetic oviduct fluid, SOF), sera (foetal calf serum, FCS vs. human serum, HS), gas atmosphere (5% CO2 in air vs. 5% CO2, 5% O2 and 90% N2) and coculture with bovine oviduct epithelial cells (cells vs. no cells) on the in-vitro development of in-vitro matured and fertilized bovine oocytes were examined. Immature oocytes surrounded with compacted cumulus cells were cultured for 24 h in TCM199 supplemented with 10% FCS, 10 micrograms follicle-stimulating hormone (FSH)/ml and 10 micrograms luteinizing hormone (LH)/ml, 1 microgram oestradiol/ml, and 1 x 10(6) granulosa cells at 39 degrees C under 5% CO2 in air. In-vitro fertilization was performed with frozen-thawed, heparin-treated (100 micrograms/ml, 15 min) spermatozoa from 2 bulls. Oocytes were incubated with 2.5 x 10(6) spermatozoa/ml for 24 h and then cultured in one of 16 treatments for 7 days. Cleavage (2-8-cell) and development to blastocysts were recorded on Days 2 and 7, respectively, after the start of culture. SOF was superior to TCM199 for cleavage (P less than 0.01), development to blastocysts (P less than 0.001) and for proportion of cultured ova resulting in blastocysts with at least 60 or at least 100 nuclei (P less than 0.001). FCS was superior to HS for development to blastocysts (P less than 0.001) and 5% oxygen was superior to air for the proportion of ova reaching at least 60 cells (P less than 0.01). For cleavage and development to blastocysts, there was an interaction between serum and cells (P less than 0.01). In the presence of cells, ova preferred FCS, in their absence, serum had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes

The ovary of cattle contains thousands of oocytes which are enclosed primarily in the preantral follicles. Methods of culturing preantral follicles are now being developed. The aim of this study was to investigate the effect of the size of bovine preantral and early antral follicles and culture media on their in vitro growth. Individual follicles isolated by microdissection of the ovarian slices were sorted into the following size classes: 75 to 124, 125 to 174, 175 to 224, 225 to 274, 275 to 324 and > or = 325 microns. The follicles were cultured individually in TCM 199 + fetal calf serum (FCS) + supplements (FSH, estradiol-17 beta, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine and hypoxanthine) or in Menezo B2 + FCS + supplements (Experiment 1) and in TCM 199 + steer serum (SS) with or without additional supplements (Experiment 2). The total number of isolated follicles of different size classes was similar in heifers and cows. No significant difference in the growth rate of follicles of different sizes was seen in the 2 media (TC 199 and B2). However, the culture of follicles in the TCM 199 that was supplemented only with SS and contained no other additives significantly reduced follicular survival and growth in comparison with follicles cultured in the supplemented medium. The survival time of follicles was related to their initial size at the beginning of culture. The longest period of growth was for follicles 275 to 324 microns in diameter (i.e., 10.7 +/- 5.7; 12.1 +/- 6.2 and 12.2 +/- 2.7 d, respectively, for culture in supplemented Menezo B2, TCM 199 + FCS and TCM 199 + SS). Survival and growth of some follicles was maintained for 23 d.     

Effect of follicle size and quality on the ability of follicular fluid to support cytoplasmic maturation of bovine oocytes

Recent studies have shown that the developmental capacity of in vitro-matured oocytes is affected by the origin of follicular fluid (FF) supplemented to the maturation medium. The aims of this study were (1) to determine if follicle size and quality would influence the capacity of FF to support bovine oocyte maturation and (2) to determine if fetal calf serum (FCS) and FF had an additive effect when added together to the maturation medium. Follicular fluid collected from 108 follicles was classified according to size (<6, 6–8, >8 mm in diameter) and quality (healthy, early atretic, and atretic). Quality, first determined by mitosis/pycnosis ratios in granulosa cell smears, was subsequently confirmed by insulin-like growth factor binding protein (IGFBP) patterns and estradiol concentrations. While most small- or medium-sized follicles showed some atresia (88% and 67%, respectively), fewer of the large follicles were atretic (30%). In experiment 1 bovine oocytes (n = 2,152) were matured either in TCM199 alone, with 10% FCS, or with 10% FF from the following follicle types: small healthy (SH); small early atretic (SEA); small atretic (SA); medium healthy (MH); medium early atretic (MEA); medium atretic (MA); large healthy (LH); large early atretic (LEA); and large atretic (LA). Following IVM, oocytes were fertilized and subsequently cultured in synthetic oviduct fluid (SOF). Day 8 blastocyst yields were 23% in TCM199 alone; 37% in TCM199 plus FCS; and, in medium supplemented with FF, SH, 36%; MH, 32%; LH, 30%; SEA, 21%; MEA, 26%; LEA, 28%; SA, 32%; MA, 33%; and LA, 38%. All FF from healthy or atretic follicles resulted in significantly improved blastocyst yields compared to M199 alone (P < 0.05) However, FF from early atretic follicles irrespective of size did not yield a significant improvement. In experiment 2 we examined the effect of addition of FF-LH and serum together to the maturation medium. In terms of blastocyst yield, no additional benefit was observed when TCM199 was supplemented with 10% FCS + 10% FF (33%) compared to 10% FCS or FF alone (35% and 30%, respectively). The efficacy of FF as a supplement to the maturation medium to improve cytoplasmic maturation appears to vary with follicle quality but not size. However, in general, the addition of 10% FF or FCS to the maturation media resulted in a similar blastocyst yield with no additional improvement when media was supplemented with both FCS and FF. © 1996 Wiley-Liss, Inc.     

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (T_g′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the T_g′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer T_g′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of T_g′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both T_g′ of freeze-drying buffer and temperature during the drying phase.     

Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection

Successful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1-4) did not increase, even at 180 min (0.7-4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120-180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0-60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39. The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.     

The use of the acridine orange test and the TUNEL assay to assess the integrity of freeze-dried bovine spermatozoa DNA

The ability to detect nuclear damage is an important tool for the development of sperm preservation methods. We used the acridine orange test (AOT) and the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) assay to assess the DNA status of sperm cells preserved with different lyophilization media. The AOT did not detect any differences between different lyophilization media. However, differences in DNA integrity were observed among treatments with the TUNEL assay, suggesting that TUNEL is a more sensitive method to evaluate sperm DNA. The use of TCM 199 and 10% FCS as a lyophilization medium resulted in 14% of the cells with DNA fragmentation in TUNEL test. The AOT indicated only 4% of the cells with chromatin damage, with this same treatment, with no significant differences when compared to the other treatments. The degree of DNA fragmentation was negatively related to fertilizing potential, as sperm DNA damage was inversely correlated with pro-nucleus formation. The TUNEL assay was found to be an efficient method to detect DNA damage in sperm, and it could be used as a tool to predict male fertility.     

Effects of epidermal growth factor on maturation, fertilization and development of bovine follicular oocytes

When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 0 to 50 ng/ml EGF, the rate of metaphase II oocytes of 30 ng/ml EGF (97%) was significantly (P < 0.05) higher than that of the control (77%), 10 (85%), and 50 ng/ml EGF (82%). After in vitro fertilization, the rate of monospermic oocytes of 30 ng/ml (75%) and 50 ng/ml EGF (77%) was significantly (P < 0.05) higher than that of the control (56 %). When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 30 ng/ml EGF and/or 10% FCS and fertilized with frozen-thawed spermatozoa, the rate of monospermic oocytes was significantly (P < 0.05) higher in EGF + FCS (82%) than in EGF (61%) and FCS (67%). The rate of oocytes with 2 pronuclei was significantly (P < 0.05) higher in EGF + FCS (54%) than in EGF (27%). When in vitro-fertilized bovine embryos were cultured for 8 d with granulosa cells in TCM 199 containing 0, 10, 30 and 50 ng/ml EGF, the rate of embryos developing to the blastocyst stage was not significantly different among the control (22%), 10 ng/ml (20%), 30 ng/ml (18%), and 50 ng/ml (20%) EGF groups. These results indicate that EGF has a beneficial effect on in vitro maturation and fertilization of oocytes and that EGF plus FCS also have a beneficial effect on normal fertilization of oocytes. However, EGF had no beneficial effect on in vitro development of embryos when they were co-cultured with granulosa cells in medium with FCS.     

Mammalian freeze-dried sperm can maintain their calcium oscillation-inducing ability when microinjected into mouse eggs

Mammalian freeze-dried sperm can maintain their genetic integrity and event support full development to term when microinjected into mature oocytes. However, it is unknown whether freeze-dried sperm can still maintain their calcium oscillation-inducing capability. Here, we microinjected mouse and bovine freeze-dried sperm into mouse MII oocytes and examined their calcium oscillation-inducing ability following intracytoplasmic sperm injection (ICSI). Two pieces of information are revealed. First, nearly all oocytes injected with a freeze-dried mouse sperm head or a bovine sperm showed fertilization-like calcium oscillations, indicating that freeze-drying treatment does not affect the activity of the sperm factor responsible for calcium oscillations. Second, freeze-dried sperm exhibited high resistance to external temperature increase. This is shown by the finding that the freeze-dried sperm can maintain their calcium oscillation-inducing capacity even following exposure to 100 degrees C for 3 h. We therefore conclude that mammalian sperm can maintain their calcium oscillation-inducing capability following freeze-drying, rehydration, and ICSI treatments.     

海藻糖对猪精子冷冻真空干燥保存效果的影响

猪精子经冷冻干燥后,在光学显微镜和电子显微镜下观察其超微结构,并借助辅助生殖技术将其注入猪卵母细胞后,进一步观察受精卵的发育情况。结果表明：海藻糖组雄原核形成率 (68.52％)、卵裂率 (59.17％) 和囊胚率 (19.16％) 优于EDTA组 (64.59％、56.26％和15.62％) 和对照组 (35.36％、52.33％和8.60％) (P＜0.05);海藻糖组的冷冻真空干燥猪精子分别在4℃下保存60、120、180 d,雄原核形成率、卵裂率和囊胚率均无显著差异 (P＞0.05);海藻糖组的冷冻真空干燥猪精子复水化后孵育1 h和2 h,卵裂率、卵裂率和囊胚率均差异显著 (P＜0.05);海藻糖处理组与EDTA处理组中的冷冻真空干燥猪精子分别在4℃和?20℃下保存后各处理组间精子形态差异不显著 (P＞0.05);海藻糖组中B级冷冻真空干燥精子百分数显著多于EDTA处理组 (P＜0.05)。超微结构分析表明,冷冻真空干燥猪精子的损伤主要表现在顶体和颈部的肿胀与缺损、尾部断裂。     

Equine oocyte in vitro maturation: influences of sera, time, and hormones.

Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oocyte IVM were evaluated: none, bovine luteinizing hormone (bLH; 1, 10, 100 micrograms/ml), equine luteinizing hormone (eLH; 100 micrograms/ml), bovine follicle-stimulating hormone (FSH; 5 micrograms/ml), and equine chorionic gonadotropin (eCG; 1 and 100 IU/ml). Cumulus expansion in the media and sera experiments was 50% (DM with BSA), 80% (TCM, B2, and DM with MS or MSO), and 100% (FCS with any medium). The proportion of metaphase II (MII) oocytes was significantly (P less than 0.05) increased the percentage of MII oocytes as compared with 0 hr of culture. Cumulus expansion in the hormone experiments was 80% (none, bLH, and eLH), and 100% (eCG and FSH). Freshly prepared bLH significantly (P less than 0.05) inhibited nuclear maturation of equine oocytes. In summary, 15 hr of culture was sufficient time for equine oocyte IVM and all combinations of medium, serum, and hormone addition were equally effective in achieving IVM except fresh bLH and DM with BSA.     

Low temperature preservation of mouse fetal germ cells at 4 degrees C

Mouse fetal germ cells(FGCs) isolated or within genital ridges from male fetuses at 15.5 d post coitum were preserved at 4 degrees C for 3-15 d with TCM-199 medium supplemented with 10, 50 or 100% fetal bovine serum (FCS) and 0.1, 0.6 or 1.0 M sucrose. The viability of FGCs was assessed by the dye exclusion test with trypan blue and nuclear transfer into enucleated oocytes and then into enucleated 2-cell embryos. Forty-one to 45 % of FGCs survived for 5 d after preservation in the medium containing 50% FCS and 0.1 M sucrose according to the results of the dye exclusion test. But significant good effect of sucrose was not observed regardless of its concentration. When genital ridges were preserved, 15-22% of FGCs survived even 10 d after preservation. The efficiency of the in vitro development of FGCs to blastocysts was similar when they were fused with enucleated oocytes and 2-cells blastomeres after 2 to 4 d storage(27-33%) compared with that of control FGCs(56%).     

Ability to activate oocytes and chromosome integrity of mouse spermatozoa preserved in EGTA Tris-HCl buffered solution supplemented with antioxidants

Potential methods for cryopreservation of mouse spermatozoa are freeze-drying, desiccation, and suspension in EGTA Tris-HCl buffered solution (ETBS: 50 mM NaCl, 50 mM EGTA, and 10 mM Tris-HCl). To determine the duration that mouse spermatozoa suspended in ETBS-based solutions could retain their normal characteristics without freezing, spermatozoa collected from the cauda epididymis were suspended in ETBS or in ETBS supplemented with the antioxidants, dimethyl sulfoxide (DMSO), or DL-alpha-tocopherol acetate (Vitamin E acetate; VEA) diluted in DMSO, then held at ambient temperature (22-24 degrees C) for up to 9 days. When oocytes were injected with spermatozoa preserved in ETBS alone, activation rates of oocytes and chromosome integrity at the first cleavage metaphase decreased at 1 day (P < 0.001) and 2-4 days (P < 0.01) following treatment. When oocytes were injected with spermatozoa preserved in ETBS supplemented with DMSO or VEA/DMSO, chromosome integrity did not decrease significantly (through 9 days of preservation). Although DMSO maintained sperm chromosome integrity more effectively than VEA/DMSO up to 2-4 days (91 and 67%, normal karyotypes in DMSO and VEA/DMSO, respectively), VEA/DMSO helped to maintain the ability of spermatozoa to activate oocytes, but did not enhance the maintenance of sperm chromosome integrity. These results suggested that deterioration of spermatozoa preserved in ETBS alone was delayed by supplementation with antioxidants.     

Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying

Disaccharides are well-known reagents to protect biostructures like proteins and phospholipid-based liposomes during freezing and drying. We have investigated the ability of the two disaccharides trehalose and sucrose to stabilize a novel, non-phospholipid-based liposomal adjuvant composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) upon freeze-drying. The liposomes were freeze-dried using a human dose concentration containing 2.5 mg/ml DDA and 0.5 mg/ml TDB with varying concentrations of the two sugars. The influence on particle size upon rehydration was investigated using photon correlation spectroscopy (PCS) and the gel to fluid phase transition was examined by differential scanning calorimetry (DSC). Data revealed that concentrations above 211 mM trehalose protected and preserved DDA/TDB during freeze-drying, and the liposomes were readily rehydrated. Sucrose was less efficient as a stabilizer and had to be used in concentrations above 396 mM in order to obtain the same effect. Immunization of mice with the tuberculosis vaccine candidate Ag85B-ESAT-6 in combination with the trehalose stabilized adjuvant showed that freeze-dried DDA/TDB liposomes retained their ability to stimulate both a strong cell-mediated immune response and an antibody response. These findings show that trehalose at isotonic concentrations protects cationic DDA/TDB-liposomes during freeze-drying. Since this is not the case for liposomes based on DDA solely, we suggest that the protection is facilitated via direct interaction with the headgroup of TDB and a kosmotropic effect, whereas direct interaction with DDA plays a minor role.     

Comparison of various maturation treatments on in vitro maturation of goat oocytes and their early embryonic development and cell numbers

In the present study, comparison of 2 different culture media (Ham's F-12 and M-199) for supporting in vitro maturation of goat oocytes, and their subsequent embryonic development was evaluated in the presence or absence of sera (estrous goat serum, EGS and fetal calf serum, FCS) and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol, 1 ug/ml). Neither medium (Ham's F-12 or M-199) when supplemented with EGS and hormones showed any notable changes in the maturation rate nor in cleavage and blastocyst development. The mean cell number for blastocysts was also significantly low (P < 0.05). However, Ham's F-12 medium supplemented with FCS and hormones showed a considerable increase in the maturation rate, but subsequent embryonic development was not appreciably increased. However, maturation, cleavage and blastocyst development rates of oocytes matured in M-199 medium in combination with 10% FCS and hormones were significantly higher (P < 0.05). Mean cell number per blastocyst was also significantly increased in this latter treatment compared with that of the other groups (P < 0.05). The results thus indicated that both the culture medium and serum have a marked effect on maturation and subsequent embryonic development. Further, the results also showed that the combination of M-199 with FSH, LH and E2 supplemented with 10% FCS was the most efficacious medium for in vitro maturation and subsequent embryonic development of the media, sera and hormone combinations studied.     

Importance of primary culture conditions for the development of rat ICSI embryos and long-term preservation of freeze-dried sperm

Rat sperm freeze-dried in a solution containing Tris and ethylenediaminetetraacetic acid (EDTA) (TE buffer) can be preserved at 4 °C, and oocytes injected with these sperm developed into offspring though developmental ability was low. We studied the culture conditions to improve the developmental ability of oocytes injected with freeze-dried sperm. After being injected with fresh sperm, the zygotes were cultured in modified Krebs–Ringer bicarbonate (mKRB), modified rat 1-cell embryo culture medium (mR1ECM)/BSA, and mR1ECM with different osmolality, before being cultured in mR1ECM. High proportion of zygotes cultured in mKRB (270 mOsm) before being cultured in mR1ECM developed into blastocysts compared to zygotes cultured only with mR1ECM (50% vs. 28%, P < 0.05). Culturing in mKRB also led to a high proportion of zygotes developing into blastocysts after the injection of freeze-dried sperm than zygotes cultured only with mR1ECM (32% vs. 15%, P < 0.05). Offspring (16%) were obtained when 19 2-cell embryos derived from oocytes that had been injected with freeze-dried sperm preserved at 4 °C for 1 year were transferred. This study demonstrated that the culture conditions soon after the injection of sperm markedly influenced the subsequent development of embryos. Also, rat sperm after freeze-drying in TE buffer were preserved at 4 °C for long term without their deterioration.     

Calcium concentration in vitrification medium affects the developmental competence of in vitro matured ovine oocytes

The present study was designed to determine whether different calcium concentrations in the vitrification solutions could improve the developmental competence of in vitro matured ovine oocytes after cryopreservation. In vitro matured oocytes were vitrified with 16.5% ethylene glycol (EG) + 16.5% dimethylsulfoxide (DMSO) vitrification media. The base media contain different calcium concentrations, so that five experimental groups were obtained: TCM/FCS (TCM 199 + 20% fetal calf serum (FCS), [Ca²⁺] 9.9 mg/dl); PBS/FCS (Dulbecco Phosphate Buffered Saline (PBS) + 20% FCS, [Ca²⁺] 4.4 mg/dl); PBS^{CaMg free}/FCS (PBS without Ca²⁺ and Mg²⁺ + 20% FCS [Ca²⁺] 2.2 mg/dl); PBS/BSA (PBS + 0.4% bovine serum albumin (BSA), [Ca²⁺] 3.2 mg/dl) and PBS^{CaMg free}/BSA (PBS without Ca²⁺ and Mg²⁺ +0.4% BSA, [Ca²⁺] 0.4 mg/dl). After warming, the oocytes from the five experimental groups were assessed for survival, spontaneous parthenogenetic activation and developmental capacity via in vitro fertilization. Oocyte survival after vitrification procedures was better preserved in group PBS^{CaMg free}/FCS compared to the others (P < 0.05). In addition, a positive correlation was found between calcium concentration in vitrification solutions and spontaneous parthenogenetic activation (correlation index 0,82; P < 0.001). Development of vitrified oocytes was significantly affected by vitrification media composition (P < 0.01). In particular, oocytes from group PBS^{CaMg free}/FCS led to higher cleavage rates and blastocyst rate compared to the others. Our data showed that lowering calcium concentration in the vitrification medium improves the blastocyst rate of vitrified ovine oocytes, probably reducing the effect of EG + DMSO during vitrification. On the contrary, the replacement of FCS with BSA dramatically reduces the developmental potential of these oocytes.     

Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying

Disaccharides are well-known reagents to protect biostructures like proteins and phospholipid-based liposomes during freezing and drying. We have investigated the ability of the two disaccharides trehalose and sucrose to stabilize a novel, non-phospholipid-based liposomal adjuvant composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6′-dibehenate (TDB) upon freeze-drying. The liposomes were freeze-dried using a human dose concentration containing 2.5 mg/ml DDA and 0.5 mg/ml TDB with varying concentrations of the two sugars. The influence on particle size upon rehydration was investigated using photon correlation spectroscopy (PCS) and the gel to fluid phase transition was examined by differential scanning calorimetry (DSC). Data revealed that concentrations above 211 mM trehalose protected and preserved DDA/TDB during freeze-drying, and the liposomes were readily rehydrated. Sucrose was less efficient as a stabilizer and had to be used in concentrations above 396 mM in order to obtain the same effect. Immunization of mice with the tuberculosis vaccine candidate Ag85B-ESAT-6 in combination with the trehalose stabilized adjuvant showed that freeze-dried DDA/TDB liposomes retained their ability to stimulate both a strong cell-mediated immune response and an antibody response. These findings show that trehalose at isotonic concentrations protects cationic DDA/TDB-liposomes during freeze-drying. Since this is not the case for liposomes based on DDA solely, we suggest that the protection is facilitated via direct interaction with the headgroup of TDB and a kosmotropic effect, whereas direct interaction with DDA plays a minor role.     

In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera

Media (TCM-199 and Ham's F-10); sera (fetal calf serum, FCS, and buffalo estrous serum, BES); and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol 1 ug/ml) were tested to determine the efficiency of in vitro maturation and fertilization of buffalo follicular oocytes. Immature good quality cumulus-oocyte complexes (COCs) were randomly assigned to 1 of 4 experiments. Each experiment consisted of 6 treatment groups. Oocytes cultured for 24 hours in medium (TCM-199 or Ham's F-10) containing 10% FCS or BES had a significantly higher maturation rate than those in medium alone (P < 0.05). However, the maturation rate was higher in medium supplemented with 10% FCS than with 10% BES. Addition of hormones alone or in combination with sera further improved the maturation rate, but no significant difference was observed in the maturation rate among the 3 hormone-treated groups. Immature oocytes matured in the various cultures were fertilized with frozen-thawed buffalo spermatozoa. Our findings show that hormone and/or serum supplementation of TCM-199 did not improve the fertilization rate. Supplementation of Ham's F-10 with LH alone or in combination with LH + FSH + E(2) and with FCS significantly improved the fertilization rate of oocytes while medium with FSH, E(2) or no hormones did not (P < 0.05); same media supplemented with BES resulted in lower fertilization rates both in the presence or absence of hormones. The results indicate that the culture medium has a marked effect on the fertilization rate of buffalo oocytes. Ham's F-10 + LH + FSH + E(2) supplemented with FCS was the most efficacious culture system of those studied for the in vitro maturation of buffalo oocytes.