Rat Hindlimb Cryopreservation and Transplantation: A Step Toward "Organ Banking".

In 2016, over 5 million reconstructive procedures were performed in the United States. The recent successes of clinical vascularized composite allotransplantations, hand and face transplantations included, established the tremendous potential of these life-enhancing reconstructions. Nevertheless, due to limited availability and lifelong immunosuppression, application is limited. Long-term banking of composite transplants may increase the availability of esthetically compatible parts with partial or complete HLA matching, reducing the risk of rejection and the immunosuppressive burden. The study purpose was to develop efficient protocols for the cryopreservation and transplantation of a complete rodent limb. Directional freezing is a method in which a sample is cooled at a constant-velocity linear temperature gradient, enabling precise control of the process and ice crystal formation. Vitrification is an alternative cryopreservation method in which the sample solidifies without the formation of ice crystals. Testing both methods on a rat hindlimb composite tissue transplantation model, we found reliable, reproducible, and stable ways to preserve composite tissue. We believe that with further research and development, cryopreservation may lead to composite tissue "banks." This may lead to a paradigm shift from few and far apart emergent surgeries to wide-scale, well-planned, and better-controlled elective surgeries.

Beneficial effect of directional freezing on in vitro viability of cryopreserved sheep whole ovaries and ovarian cortical slices.

STUDY QUESTION: Does directional freezing improve the structural and functional integrity of ovarian fragments compared with conventional slow freezing and to whole ovary cryopreservation? SUMMARY ANSWER: Compared with slow freezing, the use of directional freezing significantly improves all structural and functional parameters of ovarian fragments assessed in vitro and, overall, whole ovaries were better preserved than ovarian fragments. WHAT IS KNOWN ALREADY: Directional freezing has been developed to provide an alternative way to cryopreserve large biological samples and it is known to improve the structural and functional integrity of whole ovaries. Conventional slow freezing of ovarian fragments is the procedure more widely used in clinical settings but it causes substantial structural damage that limits the functional period after transfer back into the patient. STUDY DESIGN, SIZE, DURATION: We performed a 2 × 2 factorial design experiment on a total of 40 sheep ovaries, divided into four groups (n = 10 ovaries per group): (i) directional freezing of whole ovary (DFwo); (ii) directional freezing of ovarian fragments (DFof); (iii) conventional freezing of whole ovary (CFwo); (iv) conventional freezing of ovarian fragments (CFof). An additional eight ovaries were used as fresh controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ewe ovaries were randomly assigned to one of the experimental groups and frozen accordingly. Upon thawing, ovarian tissue was examined morphologically and cultured in vitro for 7 days. Samples were analyzed for cell proliferation and apoptosis, for DNA damage and repair activity, and for the presence of a panel of heat shock proteins (HSPs) by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Most studied parameters were significantly improved (P < 0.05) in all samples cryopreserved with directional compared with slow freezing. The proportion of primordial follicles, which developed to the primary stage in whole ovaries (53 ± 1.7%) and in ovarian fragments (44 ± 1.8%) cryopreserved with directional freezing, was greater than with slow frozen whole ovaries (6 ± 0.5%, P = 0.001) or fragments (32 ± 1.5%, P = 0.004). After 7 days of culture, cell proliferation in DFwo (28 ± 0.73%) was the highest of all groups (P < 0.05) followed by DFof (23 ± 0.81%), CFof (20 ± 0.79%) and CFwo (9 ± 0.85%). Directional freezing also resulted in a better preservation of the cell capacity to repair DNA damage compared with slow freezing both in whole ovaries and ovarian fragments. Apoptosis and HSP protein levels were significantly increased only in the CFwo group. Direct comparison demonstrated that, overall, DFwo had better parameters than DFof and was no different from the fresh controls. LIMITATIONS, REASONS FOR CAUTION: The study is limited to an in vitro evaluation and uses sheep ovaries, which are smaller than human ovaries and therefore may withstand the procedures better. WIDER IMPLICATIONS OF THE FINDINGS: Improved integrity of ovarian morphology may translate to improved outcomes after transplantation. Alternatively, the particularly good preservation of whole ovaries suggests they could provide a source of ovarian follicles for in vitro culture in those cases when the presence of malignant cells poses a substantial risk for the patient. STUDY FUNDING/COMPETING INTEREST(S): Supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 10376, Carraresi Foundation and by Legge 7 Regione Autonoma Sardegna (R.A.S). There are no conflicts of interest.

Directional freezing of reproductive cells and organs.

Directional freezing is based on a simple thermodynamic principle where ice crystals are precisely controlled through the sample by regulating the velocity of the sample movement through the predetermined temperature gradient. Directional freezing permits a precise and uniform cooling rate in both small and large volume samples. Directional freezing was used for slow and rapid freezing, as well as for vitrification of oocytes and embryos using the minimum drop size technique. Sperm samples from a wide range of domestic and wild animals were successfully cryopreserved using the directional freezing method. The method enabled, for the first time, successful freezing of a whole ovary and freeze-drying of mammalian cells followed by thawing and transplantation and rehydration, respectively.

Ovarian function 6 years after cryopreservation and transplantation of whole sheep ovaries.

Whole ovary cryopreservation and transplantation has been proposed as a method for preserving long-term ovarian function. This work reports ovarian function 6years post transplantation of frozen-thawed whole sheep ovaries. Three 9-month-old Assaf sheep underwent unilateral oophorectomy to provide organs for the experiments. After perfusing with cold University of Wisconsin solution supplemented with 10% dimethyl sulphoxide, ovaries were cryopreserved using unidirectional solidification freezing technology. After thawing, ovaries were re-perfused and re-transplanted orthotopically by microvascular re-anastomosis, to the contralateral ovarian pedicle after removing the remaining ovary. Six years following transplantation and after inducing superovulation, the sheep were killed and the ovaries analysed. Two ovaries had normal size and shape showing some recent corpora lutea, while the third showed atrophic changes. A total of 36 antral follicles were counted by transillumination and four germinal vesicle oocytes were aspirated and matured in vitro to metaphase II. Serum progesterone concentrations were indicative of ovulatory activity in one of the three sheep. Histological evaluations revealed normal tissue architecture, intact blood vessels and follicles at various stages. Currently, this is the longest recorded ovarian function after cryopreservation and re-transplantation. Cryopreservation of whole ovaries, using directional freezing combined with microvascular anastomosis, is a promising method for preserving long-term reproductive capacity and endocrine function.

Effects of dietary fats differing in n-6:n-3 ratio fed to high-yielding dairy cows on fatty acid composition of ovarian compartments, follicular status, and oocyte quality.

The objectives were to determine the incorporation of dietary encapsulated fats differing in n-6:n-3 ratio into milk fat, plasma, and various ovarian compartments and to examine the effects on ovarian follicular status, preovulatory follicle characteristics, and oocyte quality. Twenty-four multiparous Israeli Holstein cows, averaging 114 d in milk, were assigned to 1 of 3 treatment groups: 1) control (n=7), in which cows were fed a lactating cow diet; 2) E-FLAX (n=8), in which cows were fed a lactating cow diet that consisted of 1kg/d of encapsulated fat (3.8% of dry matter) containing 40.8% flaxseed oil, providing 242.2g of C18:3n-3 (low n-6:n-3 ratio); or 3) E-SUN (n=9), in which cows were fed a lactating cow diet that consisted of 1kg/d of encapsulated fat (3.8% of dry matter) containing 40.8% sunflower oil, providing 260.0g of C18:2n-6 (high n-6:n-3 ratio). Ovaries were monitored by ultrasonography for follicular status, and after synchronization, follicles >7mm were aspirated and evaluated. Ovum pickup was performed (19 sessions for the control and E-FLAX groups and 11 for the E-SUN group), and in vitro maturation and oocyte fertilization were conducted. The E-FLAX treatment increased the proportions of C18:3n-3 (5.8 fold), C20:5n-3, and C22:5n-3 (approximately 4-fold) in milk fat as compared with the other 2 treatments. The proportion of C18:3n-3 fatty acid in plasma increased dramatically with the E-FLAX treatment, from 1.43 and 1.49% in the control and E-SUN groups, respectively, to 7.98% in the E-FLAX group. Consequently, the n-6:n-3 ratio in plasma was reduced from approximately 42 in the control and E-SUN groups to 6.74 in the E-FLAX group. Proportions of C18:3n-3 in follicular fluid and granulosa cells were approximately 5-fold higher in the E-FLAX group than in the other 2 groups. The percentage of C18:2n-6 in cumulus-oocyte complexes of cows in the E-SUN group was 54% higher than that in the E-FLAX group and was 2.4-fold higher than that in the control group; the proportion of C18:3n-3 in the E-FLAX group was 4.73% and was not detected in the other groups. The average numbers of 2- to 5-mm follicles on d 5 and 9 of the cycle were higher in the E-FLAX group than in the E-SUN group, whereas the average numbers of follicles > or =10mm on d 5, 9, and 13 were higher in the E-SUN group than in the other 2 groups. The estrous cycles of the cows were synchronized and PGF(2alpha) was injected on d 16 to 17 of the cycle. The interval from PGF(2alpha) injection to behavioral estrus was longer in the E-FLAX group than in the E-SUN group, and the beginning of the luteal phase of the subsequent cycle was delayed. Concentrations of estradiol in follicular fluid of the preovulatory follicles were higher in the E-SUN group than in the E-FLAX group. The number of follicles aspirated by ovum pickup was higher in the E-FLAX group than in the control group, and the cleavage rate in the E-FLAX group was higher than in the control group, but not the E-SUN group. In conclusion, dietary n-3 fatty acids influenced the follicular status and increased the cleavage rate of oocytes as compared with those of control cows. These findings could be related to modifications of the fatty acid composition in plasma and ovarian compartments in response to dietary supplementation.

An analytical model for elucidating tendon tissue structure and biomechanical function from in vivo cellular confocal microscopy images.

Fibered confocal laser scanning microscopes have given us the ability to image fluorescently labeled biological structures in vivo and at exceptionally high spatial resolutions. By coupling this powerful imaging modality with classic optical elastography methods, we have developed novel techniques that allow us to assess functional mechanical integrity of soft biological tissues by measuring the movements of cells in response to externally applied mechanical loads. Using these methods we can identify minute structural defects, monitor the progression of certain skeletal tissue disease states, and track subsequent healing following therapeutic intervention in the living animal. Development of these methods using a murine Achilles tendon model has revealed that the hierarchical and composite anatomical structure of the tendon presents various technical challenges that can confound a mechanical analysis of local material properties. Specifically, interfascicle gliding can yield complex cellular motions that must be interpreted within the context of an appropriate anatomical model. In this study, we explore the various classes of cellular images that may result from fibered confocal microscopy of the murine Achilles tendon, and introduce a simple two-fascicle model to interpret the images in terms of mechanical strains within the fascicles, as well as the relative gliding between fascicles.

Protective effects of iodixanol during bovine sperm cryopreservation.

The aim of cryopreservation is to maintain cellular integrity, thereby enabling resumption of proper biological functioning after thawing. Here we propose OptiPrep (60% iodixanol in water) as a protectant during sperm cryopreservation using pooled bull semen as the model. We evaluated OptiPrep concentration effect and its relation to cryopreservation by comparing frozen-thawed and chilled samples. Semen, extended in Andromed with 0 (control), 1.25%, 2.5%, and 5% OptiPrep, was compared after either chilling or freezing in large volume by directional freezing. Sample evaluation included sperm motility upon thawing and after 3h incubation at 37 degrees C for frozen-thawed samples and after 3h and 6h of chilling for chilled samples; viability, acrosomal integrity, and hypoosmotic swelling were also tested for frozen-thawed and chilled samples. Chilled samples with 5% OptiPrep showed inferior viability (P=0.047) and 3h motility (P=0.017) relative to that for chilled samples with 2.5% OptiPrep and inferior viability (P=0.042), acrosomal integrity (P=0.045), and 0h motility (P=0.024) relative to that for chilled samples with 1.25% OptiPrep. The 1.25%, 2.5%, and control samples did not differ. In frozen-thawed samples, 2.5% OptiPrep was superior to all other concentrations for 3h motility (control, P=0.007; 5% OptiPrep, P=0.005; 1.25% OptiPrep, P=0.004) and to 1.25% OptiPrep for acrosomal integrity (P=0.001). In a search for a protection mechanism, we measured glass transition temperature (T(g)) of Andromed and of Andromed with 1.25%, 2.5%, and 5% OptiPrep. Andromed (-58.78 degrees C) and 1.25% OptiPrep (-58.75 degrees C) groups had lower mean T(g) than that of the 2.5% (-57.67 degrees C) and the 5% (-57.10 degrees C) groups. Directional cryomicroscopy revealed that the presence of iodixanol alters ice crystal formation into an intricate net of dendrites. Thus, iodixanol appears to possess cryoprotective properties by helping spermatozoa maintain motility and membrane integrity, possibly through altering ice crystals formation into a more hospitable environment and increasing the glass transition temperature.

Comparison of oocyte developmental competence and follicular steroid content of nulliparous heifers and cows at different stages of lactation.

Reduced reproductive performance and lower conception rates of lactating cows are closely associated with genetic progress for high milk production. In contrast, the fertility of nulliparous Holstein heifers has remained fairly stable over the years and appears to be markedly higher than that of mature lactating cows. Possible differences in oocyte quality and follicular steroid levels, which could be associated with the low fertility of high-lactating cows, were examined in 13-month-old heifers, cows around the time of first AI (60-95 d post-partum, yielding 49+/-2.4 kg/d) and cows at mid-lactation (120-225 d post-partum, yielding 37+/-2.1 kg/d). Estrus was synchronized by two doses of PGF2alpha and follicles (5-8 mm) were aspirated on days 4, 8, 11 and 15 of the cycle by an ultrasound-guided procedure. Oocytes were morphologically examined, matured in vitro, chemically activated and cultured for 8d. Cleavage rate and the proportion of developing parthenogenetic blastocysts were determined on days 3 and 8 post-activation, respectively. On day 17, heifers and cows received additional PGF2alpha and follicular fluids from preovulatory follicles were collected on day 19 perior to the expected estrus. Follicular-fluid volumes were similar in cows and heifers, as were estradiol, progesterone and androstenedione concentrations in the follicular fluid. Percentages of high-grade oocytes, proportions of cleaved oocytes and developed blastocysts did not differ between the groups. Results suggest that the fertility gap between nulliparous heifers and high-lactating cows is not directly related to steroid content in the preovulatory follicular fluid or oocyte developmental competence.

The antioxidant epigallocatechin gallate (EGCG) moderates the deleterious effects of maternal hyperthermia on follicle-enclosed oocytes in mice.

Hyperthermia-induced oxidative stress is one of the mechanisms suggested to underlie loss of developmental competence in mouse embryos. In this study, we examined whether pretreatment with the antioxidant epigallocatechin gallate (EGCG) can alleviate the negative effects of hyperthermia on developmental competence of the ovarian pool of oocytes and improve embryonic development. Female mice (CB6F1) were synchronized (eCG+hCG) and injected with 0.4 ml EGCG (100 mg/kg body weight) or with saline. Both EGCG- and saline-treated mice were exposed to heat stress (HS; 40 degrees C, 65% RH) or kept under normothermal conditions (Control; 22 degrees C, 45% RH). In vivo-derived zygotes were recovered 20 h after hCG administration and cultured in vitro. Maternal hyperthermia attenuated embryonic cleavage rate in association with further disruption in embryonic early cleavage and subsequently, with embryonic development. While pretreatment with EGCG did not affect the proportion of zygotes that cleaved to the two-cell stage, it appeared to moderate the effect of hyperthermia on both cleavage timing and developmental rate, as reflected by an increased rate of early cleaved embryos and blastocyst formation. Blastocyst developmental competence was also improved, as indicated by the increased total cell number and percentage of embryos that underwent hatching, in association with reduced apoptotic status, as reflected by the percentage of TUNEL-positive cells and intensity of caspase activity for the HS-EGCG embryos vs. HS-saline ones. In summary, while hyperthermia disrupts the competence of the follicle-enclosed oocyte, in vivo administration of the antioxidant EGCG improves developmental competence and the quality of the embryos that develop from these oocytes.

Nuclear transfer of freeze-dried somatic cells into enucleated sheep oocytes.

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.

Measurement of essential physical properties of vitrification solutions.

Vitrification is an "ice-free" cryopreservation method that has rapidly developed in recent years and might become the method of choice for oocyte cryopreservation. Five sources of damage should be considered when attempting to achieve successful oocyte cryopreservation by vitrification: (1) Solution effects (2) Crystallization (3) Glass fractures (4) Devitrification and recrystallization (5) Chilling injury. The probability of successful vitrification depends on three major factors: viscosity of the sample; cooling and warming rates; and sample volume. One of the problems associated with the vitrification solution is that it may contain high concentrations of cryoprotectants (CP), which can damage the cells through chemical toxicity and osmotic shock. In the present study, we examined the principal parameters associated with successful vitrification, and attempted to compose guidelines to the most important aspects of the vitrification process. The first step was the selection of a suitable and least toxic vitrification solution. We then evaluated the effects of cooling rate and volume on the probability of vitrification. Reduction of the sample volume, combined with accelerated cooling, enabled reduction of the CP concentration. However, in practice, a delicate balance must be maintained among all the factors that affect the probability of vitrification in order to prevent crystallization, devitrification, recrystallization, glass fractures and chilling injury.

Maternal hyperthermia disrupts developmental competence of follicle-enclosed oocytes: in vivo and ex vivo studies in mice.

Mammalian oocytes are susceptible to thermal stress at various stages of follicular development. We examined whether the ovarian pool of oocytes is susceptible to maternal hyperthermia and if so, whether hyperthermia at the germinal vesicle (GV) stage further affects the developmental competence of preimplantation embryos and offspring quality. Synchronized female mice were exposed to thermal stress (40 degrees C, 65% RH) for 1.5-2h or maintained under normothermal conditions (25 degrees C, 45% RH). Thereafter, mice were paired with stud males. In the first experiment, mated mice were sacrificed 20h post hCG administration, and in vivo-derived zygotes were recovered and cultured in vitro. Maternal hyperthermia decreased the percentage of putative zygotes of apparent normal morphology in the heat-stressed group (81+/-1.3%) as compared to the control group (86+/-1.2%). Developmental competence was also compromised as expressed by the disruption in cleavage timing pattern, resulting in a reduced developmental rate to the blastocyst stage (57+/-2.6% versus 84+/-1.9%). In the second experiment, both groups were left with stud males until litter delivery. Litter size in the first delivery cycle was lower for the heat-stressed group (7.7+/-1.1 pups), followed by a slight increase throughout consecutive cycles as compared to the control group (11.3+/-1.0 pups). Behavioral examinations of 8-week-old pups revealed similar locomotor activity and learning potential between the groups. In summary, the findings indicate that a subpopulation of the ovarian pool of follicles is highly sensitive to thermal stress and that maternal hyperthermia disrupts developmental competence of GV-stage oocytes. Pups that developed from oocytes that survived thermal stress exhibited a developmental potential similar to that of the of control pups.

Successful pregnancies with directional freezing of large volume buck semen.

Artificial insemination with frozen-thawed buck semen shows variable results which depend on many factors related to semen quality and the cryopreservation processing. We conducted experiments based on a new freezing method, directional freezing, of large volumes (8 ml). In the first experiment semen from three Saanen bucks, ages 1-2-years-old and genetically selected for milk improvement, was frozen individually. Two to three-years-old Saanen females (n = 164) were synchronized with controlled internal drug release (CIDR), pregnant mare serum gonadotrophin (PMSG) and prostaglandin. Double cervical inseminations were performed with frozen-thawed semen and fresh semen as control. In the second experiment we used pooled, washed frozen semen to examine the effect of washed seminal plasma. The motility after washing was 80-90% and after thawing was 55-65% for all bucks. The sperm concentration increased with the collections and the advance into the breeding season from 1.9 x 10(9) to 4.4 x 10(9) cell/ml average. Two inseminations were carried out at 8h intervals. The first insemination was performed at 32 h after CIDR withdrawal with fresh and frozen-thawed semen. Pregnancy rates were assessed by ultrasonography conducted 40 and 90 days post-insemination (from three bucks). Results were 58, 67, 50% with fresh semen, and for frozen semen were 33, 37 and 53%; these results were significantly different in one of the three bucks (P < 0.005). In the second experiment with pooled, washed semen the pregnancy rate was 41.6%, which compared with the average results of the frozen semen in the first experiment 38.9% no significant difference was found. We conclude that freezing buck semen in large volumes (8 ml) is possible. Cryobanking of buck semen will facilitate a genetic breeding program in goats and preservation of biodiversity. Washed semen did not improve the fertility of the semen when Andromed bull extender is used.

Transillumination increases oocyte recovery from ovaries collected at slaughter. A new technique report.

An inexpensive and convenient method of collecting large number of oocytes for in vitro procedures is by aspiration of follicles visible on the surface of isolated ovary. This method yielded only moderate numbers of oocytes per ovary, and it was found that the yield could be improved by slicing the tissue to reach deep, cortical follicles. However, slicing was time consuming and increased chances for sepsis. We developed a new technique that allows direct viewing of cortical follicles for aspiration of oocytes by transillumination of the ovarian medulla and cortex with a Plexiglas rod inserted through a small incision at the hilus. The technique, called "Transillumination-Aspiration Ovary" (TAO), increased the oocyte yield by 50% per ovary. The oocytes are probably recovered from deeper follicles which are difficult to identify during regular oocyte aspiration. The oocytes had a normal grading and exhibited normal in vitro development efficiency. Using the "TAO" technique we recovered 777 oocytes from 2160 follicles in 106 ovaries, a recovery rate of 36% from follicles and a mean of 7.3 oocytes/ovary. When we aspirated only surface follicles, we obtained 523 oocytes in 1384 visible follicles in 107 ovaries, for a recovery rates of 37% but a mean yield of 4.9 oocytes per ovary. Mean number of follicles were 20.5% with TAO and 12.8% without, thus recovery rates of oocytes per follicle were similar with both methods, but yield of oocytes per ovary was higher with TAO, thus showing that the difference between the two methods lies in higher numbers of visible follicles with TAO. Moreover, with the TAO technique 71% of the total oocytes we recovered (n=551) were grade I or II oocytes, in which 52% cleaved to the 2 to 4-cell stage and 26% had reached the blastocyst stage. We conclude that the method is effective for accurately locating cortical and peripheral follicles that contain oocytes suitable for IVF and in vitro embryo production (IVP).

Number of oocytes obtained from cows by OPU in early, but not late lactation increased with plasma insulin and estradiol concentrations and expression of mRNA of the FSH receptor in granulosa cells.

The effects of lactation stage and hormonal profile on the quality and quantity of oocytes and the gonadotrophic sensitivity of granulosa cells (GC) from small antral follicles obtained by sequential aspirations from ovaries of high producing dairy cows were examined. Cows in late lactation (263(+/- 60) days postpartum) and 98(+/- 16) days pregnant in positive energy balance (EB) showed no significant changes in plasma concentrations of estradiol, progesterone, insulin, IGF1 or expression of mRNA of the FSH receptor in GC from small antral follicles during the 49 days experimental period. There were no changes in the number and quality of oocytes obtained from each aspiration. In cows in early lactation (72.8 +/- 6 days postpartum), plasma insulin concentrations increased and were positively correlated with plasma estradiol concentration. Due to the sequential aspirations progesterone blood concentrations were low in early lactation cows. Expression of mRNA of the FSH receptor increased in GC from small antral follicles of early lactation cows together with the number of oocytes obtained with aspiration sessions. No differences were found in morphological quality or function between oocytes obtained from small antral follicles from cows in early or late lactation. In early, but not late lactation, the number of oocytes was correlated with both insulin and E2 plasma concentrations. Improved EB and sensitivity of GC to FSH may be involved in oocyte recruitment in early lactation.

Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo.

Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 degrees C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls (18 21 ); BSA (9 15 ); WF at 1 mg/ml (14 15 ); WF at 10 mg/ml (13 15 ) or OP at I mg/n-d (15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF (27 30 ) than for OP (26 41 ) or BSA (9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 degrees or 4 degrees C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 degrees C (WF/4 degrees C) had comparable hatching rates (8 12 ) to that of controls (10 10 ), but embryos in the other treatments (WF 0 degrees C, 5 11 , BSA 4 degrees C, 6 11 and BSA 0 degrees C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 degrees C, but embryos stored in WF 0 degrees C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 degrees C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 degrees C/min; T2, WF > 2 degrees C/min and T3, WF < 1 degrees C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 degrees C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.

Chilling sensitivity in zebrafish (Brachydanio rerio) oocytes is related to lipid phase transition.

Oocytes of zebrafish were used to study chilling sensitivity and membrane lipid phase transitions in tropical fish. The oocytes were divided into two groups: small (without yolk, <0.1mm) and large (with yolk, >0.1mm). After exposure of the oocytes to different temperatures (25, 22, 19, 16, 12, 8, 0, -8+0.5 degree C) for 15 minutes, the integrity of their membranes was determined by carboxyfluorescein diacetate (CFDA) staining. At 16 and 12 degree C, damage was maximum (membrane integrity decreased by 50%) for small and large sizes, respectively. Lipid phase transition (LPT), which was evaluated using Fourier transform infrared (FTIR) microscopy, indicated phase transitions at the same temperatures at which damage was maximal (between 22 and 12 degree C).In another series of experiments, the chilling sensitivity of oocytes taken from zebrafish which had been held at 16 degree C for different periods of time (0, 15, 30, 60 minutes) was determined as described above. In small oocytes membrane integrity decreased after 15 minutes, and in large oocytes integrity decreased after 30 minutes. Chilling sensitivity was also measured in oocytes from zebrafish that had been held at 16 degree C for 30 minutes and then rewarmed to 28 degree C for 2 hours. Despite this recovery period, the integrity of the oocytes remained low. We suggest that chilling sensitivity in zebrafish oocytes is related to lipid phase transition of their membranes and starts at 10 degree C below the physiological temperature

Does membrane lipid profile explain chilling sensitivity and membrane lipid phase transition of spermatozoa and oocytes?

Ram, fowl and bee spermatozoa, and oocytes of cows and zebrafish were used to study lipid membrane profiles, chilling sensitivity and lipid-phase transitions. The integrity of the membranes was determined by carboxyfluorescein diacetate (CFDA) staining following exposure for 15 minutes to low temperatures. Ram and fowl spermatozoa showed different degrees of loss of membrane integrity. Surprisingly, bee spermatozoa did not show any sensitivity to chilling, and their membranes remained intact down to 0 degree C. In bovine oocytes (at the GV stage) chilling injury was very severe at 16 degree C (membrane integrity decreased by 50%). Lipid phase transition (LPT) and membrane fluidity, which were evaluated by Fourier transform infrared (FTIR) microscopy, and fluorescence polarisation, showed phase transitions at the same temperatures as caused damage (between 30 and 12 degree C). The membrane lipid profiles showed high concentrations of polyunsaturated fatty acids (PUFA) in cold-sensitive ram spermatozoa and zebrafish oocytes, but the ratio between PUFA and saturated fatty acids was highest in cold-resistant bee spermatozoa and lowest in cold-sensitive bovine oocytes. These results suggest a close relationship among cold susceptibility, lipid phase transition and lipids profile in animal gametes.

Cryopreservation of oocytes and embryos.

Two hundred years have passed since the first description of supercooled water by Gey-Lussac to the recently high survival rates of embryo and oocytes after vitrification. This review discusses important milestones that have made vitrification the method of choice for oocytes and embryos cryopreservation. We will go through the first cells ever to survive low temperature exposure in the beginning of the last century, the finding of glycerol in the late 1940s and the first mouse and bovine embryos freezing in the 1970s. During the 1980s, embryo vitrification began and the time since is a tribute to the development of oocytes vitrification. Standardization and an automatic vitrification procedure are currently under development. The next evolutionary step in oocyte and embryo cryopreservation will be preserving them in the dry state at room temperature, allowing home storage for future use a reality.