Mineral Oil for in vitro Embryo Production: What We Should Know?

Mineral oil as a barrier can minimize temperature, osmolality, and pH fluctuation of the media in the in vitro embryo production system (IVP). Regardless of these advantages, mineral oil quality is varied and may deteriorate during storage or transport conditions. So, it can affect the IVP outcome by absorbing the essentials factors or realizing the toxic components into the media. Although, some methods have already been developed to reduce these side effects, still there is a big concern about the safety and use of mineral oil in the IVP system. In this review, we provided an overview of the advantages and disadvantages of using mineral oil in the IVP system. We also reviewed available methods for its quality control and finally, we introduced some methods for reducing the side effects of mineral oil.

The effect of parity number on the metabolism, inflammation, and oxidative status of dairy sheep during the transition period.

The objective of this study was to evaluate whether dairy sheep during the transition period are affected by their parity numbers with regard to (1) body weight (BW), body condition score (BCS), and production performance (milk yield and composition) and (2) metabolic, inflammation, and stress biomarkers. For this purpose, 30 Sarda dairy ewes [15 primiparous (PRP) and 15 multiparous (MUP) ewes] were recruited on d 90 of gestation. Each group was homogeneous according to age, BW, and BCS. Sampling was carried out at -60, -30, -7, 0, +30, and +60 d from lambing. The MUP ewes showed a higher BW (46.32 vs. 38.71 kg) and larger litter size (1.45 vs. 1.06 kg) but a lower BCS (2.47 vs. 2.70) than the PRP ewes. Furthermore, the MUP ewes had lower concentrations of glucose (3.49 vs. 4.27 mol/L), cholesterol (1.63 vs. 1.81 mmol/L), free fatty acids (0.47 vs. 0.62 mmol/L), and triglycerides (0.22 vs. 0.25 mmol/L) compared with PRP ewes. With regard to inflammation and oxidative stress parameters, the PRP group had higher haptoglobin (0.48 vs. 0.18 g/L) and paraoxonase (187.90 vs. 152.11 U/L) activity than the MUP group. Overall, the MUP ewes were characterized by greater milk production performance and greater feed intake, resulting in a better energy balance, than the PRP ewes. Interestingly, these findings highlighted a different metabolic and inflammatory response over the transition period between PRP and MUP ewes, with the latter displaying lower concentrations of inflammatory-related biomarkers.

Changes in renal hemodynamics of undernourished fetuses appear earlier than IUGR evidences.

The present study used a sheep model of intrauterine growth restriction, combining maternal undernutrition and twinning, to determine possible markers of early damage to the fetal kidney. The occurrence of early deviations in fetal hemodynamics which may be indicative of changes in blood perfusion was assessed by Doppler ultrasonography. A total of 24 sheep divided in two groups were fed with the same standard grain-based diet but fulfilling either their daily maintenance requirements for pregnancy (control group; n=12, six singleton and six twin pregnancies) or only the 50% of such quantity (food-restricted group; n=12; four singleton and eight twin pregnancies). All the fetuses were assessed by both B-mode and Doppler ultrasonography at Day 115 of pregnancy. Fetal blood supply was affected by maternal undernutrition, although there were still no evidences of brain-sparing excepting in fetuses at greatest challenge (twins in underfed pregnancies). However, there were early changes in the blood supply to the kidneys of underfed fetuses and underfed twins evidenced decreases in kidney size.

High-milking sheep have a lower ovulation rate and tend to yield fewer embryos in response to superovulation and intrauterine artificial insemination.

Antagonistic relationship between milk yield and reproduction is reported in several livestock species. This study aimed to investigate whether genetic merit for milk production in dairy sheep affects responses to superovulation, embryo yield and quality. A total of 21 cross-bred Sarda x Lacaune ewes homogeneous for age, parity and stage of lactation were included. The ewes were stratified as high-producing or low-producing based on their genetic merit for milk production estimated by a pentatrait repeatability animal model. Oestrus was synchronized using an intravaginal progesterone pessary inserted on Day 0 and removed on Day 14. Superovulatory treatment consisted of 350 I.U. of porcine FSH administered in eight decreasing intramuscular doses every 12 hr with a total dose of 10 ml of solution starting 12 days after insertion of sponges. Laparoscopic artificial insemination (AI) was performed 48 hr after pessary removal. Surgical embryo recovery was performed at Day 8 after pessary removal. Correlation between breeding value for milk production and the number of corpora lutea (CL) was significantly different from zero (-0.49). High-producing ewes had a lower number of CL than low-producing counterparts (7.6 ± 2.50 vs 12.1 ± 5.16 respectively; p < .02). Furthermore, there was a tendency for high-producing ewes to yield fewer embryos than low-producing females (5.3 ± 3.46 vs 9.18 ± 5.11; p = .09). No differences were observed between ewes in both genetic groups with regard to the number of embryos of grades 1, 2 and 3. To our knowledge, this is the first report highlighting an antagonism between genetic merit for milk production and the ability to produce embryos in sheep. These results deserve to be considered in sheep breeding programmes.

Glucogenic treatment creates an optimal metabolic milieu for the conception period in ewes.

This study determined the influence of a short-term glucogenic nutritional treatment on circulating concentrations of glucose, insulin, insulin-like growth factor 1 (IGF-1), nonesterified fatty acids (NEFA), and urea, and on their correspondent levels in follicular fluid (FF) collected 12 h after the end of the treatment. After estrous synchronization with intravaginal progestagen-impregnated sponges, 20 Sarda ewes were randomly allocated into two experimental groups (GLU and WAT) and, from day 7 to day 10 (day 0 = day of sponge removal), the GLU group was gavaged with a glycogenic mixture, whereas the WAT group was gavaged with water (control group). Follicular development was stimulated by FSH administration from day 8 to 10. At day 11, ovaries were collected and follicular fluid processed. Plasma changes were assessed from day 6 to 11. In GLU group, circulating concentration of glucose (P < 0.0001), insulin (P < 0.0001), and IGF-1 (P < 0.01) rose significantly, whereas NEFA and urea concentrations decreased (P < 0.0001), as compared with controls. In particular, in FF the higher glucose concentrations found in GLU ewes compared with controls (P < 0.0001) were not accompanied by any increase in insulin and IGF-1 concentrations. NEFA (P < 0.0001) and urea (P < 0.0001) were lower in FF of GLU than WAT group, although NEFA clearance in the ovary proved to be less efficient than at the systemic level. No significant difference between groups was found in FF concentrations of pregnancy-associated plasma protein A (a protease regulating the levels of free IGF-1 in follicles), glutathione, and in its total antioxidant capacity. These results suggest that glycogenic mixture administration creates a suitable follicular microenvironment for the conception period in dairy ewes.

Cryopreservation of whole ovine ovaries with pedicles as a model for human: parameters of perfusion with simultaneous saturations by cryoprotectants.

BACKGROUND: The aim of this research was to study the effectiveness of perfusion of intact ovine ovaries with different rates of perfusion and time-period elapsed between extraction of these ovaries and the beginning of perfusion. METHODS: Ovaries were perfused through the arteria ovarica (ovarian arteries) with culture medium supplemented with 5% bovine calf serum, 6% dimethyl sulfoxide, 6% ethylene glycol, 0.15M sucrose, Indian ink, and 100 IU/mL heparin at room temperature (22 degrees C). In the first cycle of experiments, ovaries (n = 96) were perfused for 60 minutes just after extraction of ovaries at the following rates of perfusion (mL/hour): 150, 100, 75, 50, 25, 12.5, and 6.3. In the second cycle of experiments, ovaries (n = 26) were perfused at a rate of 25 mL/hour for 60 minutes after extraction of ovaries and their storage at room temperature for 2, 3, 4, and 5 hours, for groups 1, 2, 3, and, 4, respectively. Successful perfusion of blood vessels was detected visible by a blue coloration of the ovarian tissues. RESULTS: The first cycle of experiments showed that the optimal perfusion rates were 50 mL/hour and 25 mL/hour. In the second cycle of experiments, good perfusion of ovaries was established at a perfusion rate of 25 mL/hour for the ovaries of groups when 2 and 3 hours had elapsed after extraction. CONCLUSIONS: Effective perfusion of ovine intact ovaries with vascular pedicle was established using freezing medium at room temperature at the rate of perfusion of 25 mL/hour or 50 mL/hour. The ovaries must be perfused not later than 3 hours after the death of animals.

Different in vitro culture systems affect the birth weight of lambs from vitrified ovine embryos.

It has been reported that different in vitro culture systems affect the birth weight of lambs. The aim of this study was to test body weight and lambing rate of lambs born from five different in vitro culture systems after vitrification. Oocytes of Sarda sheep were matured in TCM-199 plus 0.4% bovine serum albumin (BSA) using systems: (i) 4 mg/ml fatty acid-free BSA (BSA4); (ii) 8 mg/ml fatty acid-free BSA (BSA8); (iii) BSA8-hyaluronan (BSA8-HA); (iv) BSA8-charcoal-stripped FBS (BSA8-CH); or (v) with 10% fetal bovine serum (FBS; serum) and fertilized with fresh semen. The presumptive zygotes were cultured up to the blastocyst stage with BSA8, BSA8-HA, BSA8-CH or serum or BSA4. In the third and fifth days of culture 5% charcoal-stripped FBS was added into BSA8-CH and serum, while 8 mg/ml or 4 mg/ml fatty acid-free BSA was added as BSA8, BSA8-HA and BSA4 respectively; 6 mg/ml HA was added to BSA8-HA. In total, 240 vitrified blastocysts were transferred into synchronized ewes. The lambing rate was not significant different between BSA groups or between serum groups (BSA8-CH and serum), while serum groups showed significant lower values when compared with BSA groups. Only BSA8 groups produced heavy lambs (≥4.5 kg) with a significant difference between BSA4 and BSA8 groups (P < 0.05).

Blastocyst rate of in vitro embryo production in sheep is affected by season.

It has been reported that the number and quality of in vitro produced embryos is season related. This study was conducted to assess the effect of season on cleavage, blastocyst and lambing rates of in vitro produced ovine embryos during 3 years of collection data. Ovaries of Sarda sheep were collected from a slaughterhouse. In total, 5035 oocytes were recovered and matured in TCM-199 with 4 mg/ml bovine serum albumin (BSA), 100 µM cysteamine, 0.3 mM Na pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH), 0.1 UI/ml recombinant luteinising hormone (r-LH), and 1 µg/ml estradiol-17ß. Matured oocytes were fertilized with fresh semen in synthetic oviductal fluid (SOF) with 20% heat inactivated estrous sheep serum. The presumptive zygotes were cultured for 6-7 days (blastocyst stage) in SOF medium supplemented with 1% Basel Medium Eagle (BME), 1% Minimum Essential Medium, 1 mM glutamine and 8 mg/ml fatty acid-free BSA. The embryos produced were vitrified and a total of 165 blastocysts (80 from the breeding season and 85 from the anoestrous season) were transferred in pairs into recipient ewes during the reproductive period. There were no significant differences in cleavage rates between seasons in any of the 3 years examined (84% versus 83%, 81% versus 80% and 80% versus 79%, respectively). The blastocyst rate varied significantly between seasons in 2005 and 2007 (P < 0.05), and in 2006 (P < 0.001). There were no differences in pregnancy and lambing rates between embryos during anoestrous versus during the breeding season. In conclusion, only the blastocyst rate appeared to have been affected by season, possibly due to variation in the number of developmentally competent oocytes.

Cryobanking of farm animal gametes and embryos as a means of conserving livestock genetics.

In the last few decades, farm animal genetic diversity has rapidly declined, mainly due to changing market demands and intensification of agriculture. But, since the removal of single species can affect the functioning of global ecosystems, it is in the interest of international community to conserve the livestock genetics and to maintain biodiversity. Increasing awareness on the reduction of breed diversity has prompted global efforts for conservation of farm animal breeds. The goals of conservation are to keep genetic variation as gene combinations in a reversible form and to keep specific genes of interest. For this purpose two types of strategies are usually proposed: in situ and ex situ conservation. In situ conservation is the breed maintaining within the livestock production system, in its environment through the enhancement of its production characteristics. Ex situ in vivo conservation is the safeguard of live animals in zoos, wildlife parks, experimental farms or other specialized centres. Ex situ in vitro conservation is the preservation of genetic material in haploid form (semen and oocytes), diploid (embryos) or DNA sequences. In the last few years, ex situ in vitro conservation programs of livestock genetic resources have focused interest on cryopreservation of gametes, embryos and somatic cells as well as testis and ovarian tissues, effectively lengthening the genetic lifespan of individuals in a breeding program even after the death. However, although significant progress has been made in semen, oocytes and embryo cryopreservation of several domestic species, a standardized procedure has not been established yet. The aim of the present review is to describe the cryobanking purposes, the collection goals, the type of genetic material to store and the reproductive biotechnologies utilized for the cryopreservation of farm animal gametes and embryos.

Good quality sheep embryos produced by superovulation treatment without the use of progesterone devices.

Multiple ovulation and embryo transfer (MOET) is a very important tool for the genetic improvement and preservation of endangered livestock. However, the success of a MOET programme highly depends on the number of transferable embryos in response to a superovulation treatment. Thus, the aim of this study was to compare the number and quality of embryos produced during natural oestrus under porcine FSH treatment without the use of progesterone devices to more traditional protocols. Forty Sarda sheep were divided into 2 groups: without sponges (WS) (n = 20) and with sponges (S) containing 40mg FGA for 12 d (n = 20) (control group); 350 I.U. of porcine FSH per sheep was administered in eight decreasing doses twice daily starting four days after estrus was detected (Day 0) in group WS and 48 h before sponge removal in group S. A single i.m. dose of 125 µg of cloprostenol was administered on Day 6 after estrus in group WS to induce luteolysis. Sheep were naturally mated 24 h after cloprostenol injection or sponge removal. Seven days after mating, an inguinal laparotomy was performed and the number of corpora lutea (CL) recorded. Embryos were recovered surgically by flushing each uterine horn. A total of 38 fresh and 22 vitrified embryos were transferred in pairs into 3 groups of recipients seven days after estrus detection: fresh embryos from group S (S-F) (n = 9), fresh embryos from group WS (WS-F) (n = 10) and vitrified embryos from group WS (WS-V) (n = 11). Data on the number of corpora lutea (CL), recovered ova and embryos (OER), and quality 1-2 and 3 embryos (EQ(1-2), EQ(3)) per ewe were analyzed by ANOVA. Recovery (RR), fertility (FR) and quality 1-2 embryo (Q(1-2)R) rates per treatment were analyzed by a Chi Square analysis. A Chi Square analysis was also applied to pregnancy rate (PR), lambing rate (LR) and twinning rate (TR) of fresh and vitrified embryos in order to analyze embryo transfer results. Among all superovulation variables analysed, results show statistically significant differences in mean number of CL/ ewe (9.3 ± 3.9 vs 7 ± 3.2), RR (67% vs 80 %) and FR (100% vs 80%) (P < 0.05) between WS and S groups respectively. There were no significant differences in PR (78%, 70% and 82%), LR (67%, 60% and 59%) and TR (71%, 71% and 44.4%) among S-F, WS-F and WS-V groups respectively. In conclusion, it is possible to produce a good number of transferable embryos during natural oestrus avoiding the use of sponges.

Effect of different diluents on goat semen fertility.

Skim milk (SM) is considered to be the most widely employed extender for goat sperm used for artificial insemination (AI). However, the fertilizing life span of sperm stored in milk or milk-based extenders does not exceed 12h. Besides some seminal plasma components, such as a protein fraction from the goat bulbourethral gland secretion (SBUIII), interacts with some milk fractions and inhibits the spermatozoa motility. The aim of this study was to prolong the survival of buck semen and its fertility. Buck ejaculates were diluted to a final concentration of 100x10(6)spermatozoa/ml with three different diluents: SM, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPOL+hyaluronic acid (TEMPOL+HA). At 7h from dilution 42 goats were inseminated with semen diluted with SM (short-term semen) while after storage for 24h, 44 and 45 goats were inseminated with semen diluted with TEMPOL and TEMPOL+HA (long-term storage), respectively. At day 50 from AI the percentages of pregnant goats were 71.4% (30/42) with SM, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, with significant differences between SM and TEMPOL+HA. The kidding rate was 66.7% (28/42) with SM diluent, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, without significant differences among treatment groups. In conclusion, it is possible to maintain good fertility in goats after AI with semen stored for 24h in TEMPOL.

Lambing rate using vitrified blastocysts is improved by culture with BSA and hyaluronan.

Fatty acid-free bovine serum albumin (BSA(FAF)) can be added to supplement medium used in the culture of sheep embryos. BSA(FAF) was able to support blastocyst and subsequent embryo development at rates equivalent to that of fetal calf serum (FCS)-supplemented medium when fresh embryos were transferred. Furthermore, culture with BSA(FAF) significantly increased development of vitrified blastocysts transferred into synchronized sheep. The addition of the glycosaminoglycan, hyaluronan (HA) to the culture medium in the third and fifth day also increased cryo-tolerance of blastocysts and in turn lambing rate was improved.

Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts.

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation.

Sexing of in vitro produced ovine embryos by duplex PCR.

The aim of this article was to develop a fast and easy duplex polymerase chain reaction (PCR) method, for sex determination of ovine in vitro produced embryos prior to implantation. We tested the approach with 107 samples of autosomal cells (oviductal sheep cells and male lamb fibroblasts), divided into three groups for each sex according to the number of cells employed (30, 5, 2, respectively). We then used the test on 21 embryos at blastocyst stage. On the same day the embryos were transferred in pairs into 11 recipient synchronized ewes. The PCR utilized two different sets of primers: the first pair recognized a bovine Y-chromosome-specific sequence (SRY), that showed 100% homology with the corresponding sequence of the ovine Y-chromosome and is amplified in males only. The second pair recognized the bovine 1.715 satellite DNA (SAT) which was amplified in all ovine samples but, when submitted to the GenBank database did not show homology with any of the reported ovine sequences. However, after sequencing, ovine amplification product showed 98% homology with the bovine specific satellite sequence. The autosomal samples were amplified with 85.0% efficiency and 91.2% accuracy, while amplification was successful with all 21 embryos (100% efficiency). Eight lambs were born and the sex as determined by PCR corresponded to the anatomical sex in seven (87.5% accuracy). These results confirm that this method can be applied in ovine breeding programs to manipulate sex ratio of offspring.

Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived.

We compare different vitrification protocols on the pregnancy and lambing rate of in vitro produced (IVP) and in vivo derived (IVD) ovine embryos. Ovine blastocysts were produced by in vitro maturation, fertilization and culture of oocytes collected from slaughtered ewes or superovulated and inseminated animals. Embryos were cryopreserved after exposure at room temperature either for 5 min in 10% glycerol (G), then for 5 min in 10% G + 20% ethylene glycol (EG), then for 30 s in 25% G + 25% EG (glycerol group), or for 3 min in 10% EG + 10% dimethyl sulphoxide (DMSO), then for 30s in 20% EG + 20% DMSO + 0.3 M sucrose (DMSO group). One group of in vitro produced embryos was cryopreserved similarly to the DMSO group, but with 0.75 M sucrose added to the vitrification solution (DMSO 0.75 group). Glycerol group embryos were then loaded into French straws or open pulled Straws (OPS) while the DMSO group embryos were all loaded into OPS and directly plunged into liquid nitrogen. Embryos were warmed with either a one step or three step process. In the one step process, embryos were placed in 0.5 M sucrose. The three-step process was a serial dilution in 0.5, 0.25 and 0.125 M sucrose. The embryos of DMSO 0.75 group were warmed directly by plunging them into tissue culture medium-199 (TCM-199) + 20% foetal bovine serum (FBS) in the absence of sucrose (direct dilution). Following these manipulations, the embryos were transferred in pairs into synchronised recipient ewes and allowed to go to term. The pregnancy and the lambing rate within each group of IVP and IVD embryos indicated that there was no statistical difference among the vitrification protocols.

Effect of recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo development and viability.

The objective of this work was to study the effect of a preparation of human recombinant gonadotrophins (r-FSH and r-LH) on the in vitro maturation (IVM) and development of sheep oocytes. In addition, the viability of fresh and vitrified blastocysts obtained after transfer was tested. Oocytes collected from slaughtered animals were divided into five different maturation groups. All groups were matured in a medium containing TCM199 with 4 mg/ml BSA, 100 microM cysteamine and 1 microg/ml estradiol-17beta. Each group was also treated with one of the following: 0.1 UI/ml r-FSH (r-FSH group), 0.1UI/ml r-LH (r-LH group), 0.1 UI/ml r-FSH and 0.1 UI/ml r-LH (r-FSH/r-LH group), 5 microg/ml FSH and 5 microg/ml LH hypophysial gonadotrophins (h-G group) as a control, or no gonadotrophins (no-G group). After in vitro fertilization with fresh ram semen, presumptive zygotes were cultured in vitro for 6-7 days and a total of 109 blastocysts were then transferred in pairs into synchronized ewes. To determine the viability of embryos after vitrification, 36 blastocysts from the r-FSH/r-LH group and 30 from the h-G group were vitrified in 10% ethylene glycol (EG) and 10% dimethylsulphoxide (DMSO) for 5min, followed by 20% EG, 20% DMSO and 0.5M Sucrose (S) for <45 s. They were loaded into open pulled straws (OPS) and plunged into LN(2). After warming, the blastocysts were transferred in pairs into synchronized ewes. The highest maturation rate was reached in the r-FSH/r-LH group (91.9%). However, no statistical difference was found when this group was compared with the h-G group (84.0%). Likewise, the cleavage rate of the r-FSH/r-LH group (81.4%) was not significantly different from that of the h-G group (82.3%). The cleavage rates of all other groups, however, were significantly lower than the r-FSH/r-LH and h-G groups. The blastocyst rate was highest in the h-G group (53.6%), and it was statistically higher than in the r-FSH/r-LH group (41.5%). The blastocyst rate was very similar between groups r-FSH and r-FSH/r-LH (42.0 and 41.5%, respectively). The lowest lambing rate (31.8%) was in the no-G group. The highest lambing rate was achieved in the r-FSH/r-LH group (66.6%). The vitrified embryos of h-G and r-FSH/r-LH groups had a very similar lambing rate (16.6% and 19.4%). In conclusion, these data provide support for the hypothesis that sheep oocytes respond to human recombinant gonadotrophins used for in vitro embryo production.

Vitrification of human spermatozoa without cryoprotectants.

The aim of our study was to investigate the effects of vitrification (cooling rate approximately 10000(C/min) without cryoprotectants on swim-up prepared human spermatozoa in comparison to standard conventional freezing with cryoprotectants. Motility, morphology, rate of viability and acrosome reaction of spermatozoa were evaluated. The described method of cryopreservation of human spermatozoa by direct plunging into liquid nitrogen slush without cryoprotectants was effective and could be recommended for routine IVF.

Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts.

Ovine blastocysts were produced by maturation, fertilization and in vitro culture (IVM/IVF/IVC) of oocytes from slaughtered adult and prepubertal ewes and collection from superovulated and inseminated adult animals. Dulbecco's PBS supplemented with 0.3 mM Na Pyruvate and 20% FCS was used as the basic cryopreservation solution. The embryos were exposed to the vitrification solution as follows: 10% glycerol (G) for 5 min, then 10% G +20% ethylene glycol (EG) for 5 min. Embryos were placed into 25% G + 25% EG in the center of 0.25- mL straws and plunged immediately into LN2. Warming was done by placing the straws into a water bath at 37 degrees C for 20 sec, and their contents were expelled into a 0.5 M sucrose solution for 3 min; the embryos were then transferred into 0.25 M and 0.125 M sucrose solution for 3 min each. Warmed blastocysts were transferred to the culture medium for 24 h. Survival was defined as the re-expansion of the blastocoele. All surviving blastocysts were transferred to synchronized recipient ewes, and the pregnancy was allowed to go to term. Of 68 vitrified in vitro produced blastocysts, 46 re-expanded (67.6%) and 10 lambs were born (14.7%). From the 62 in vivo derived and vitrified embryos, 52 re-expanded (83.8%) and 39 lambs were born (62.9%). The lambing rate of in vitro produced fresh transfer embryos was 40% (20 lambs/50 blastocysts transferred), and of the 32 in vivo derived blastocysts and transferred fresh, 26 lambs were born (81.2%). The results indicate that in vitro produced embryos can be successfully cryopreserved by vitrification.

Offspring from one-month-old lambs: studies on the developmental capability of prepubertal oocytes.

A wave of follicular growth in lamb ovaries occurs at about 4 weeks of age, generating a life-time peak in follicle numbers. In order to take advantage of the large number of oocytes available, and to substantially decrease the generation interval, embryos were derived from oocytes collected from 1-mo-old lambs. Animals were subjected to one of 3 regimes of hormonal stimulation: groups 1 and 2 were treated to obtain germinal vesicle-stage oocytes, and group 3 to produce mature metaphase II oocytes. Adult sheep stimulated by an appropriate dose of FSH served as control. The developmental ability of collected oocytes was evaluated by either in vivo or in vitro culture to the blastocyst stage after in vitro maturation and/or fertilization. Blastocysts were transferred immediately or after cryopreservation to suitable recipient sheep. In order to investigate the full developmental potential of these embryos, pregnancies were allowed to go to term. The results show significant differences (P < 0.001) between all experimental groups in blastocyst numbers produced. Embryos derived from group 1 animals produced the greatest number of blastocysts, under both in vivo (36. 7%), and in vitro (22.9%) culture systems. Group 2 gave lowest blastocyst production (5.0%), while group 3 yielded 13.2% blastocysts. The number of pregnant recipients carrying to term lamb-derived embryos was severely reduced for both in vivo- (2 of 9; 22.2%) and in vitro-cultured, fresh (3 of 10; 30.0%) and cryopreserved (1 of 6; 16.7%) lamb embryos. This study is the first report of the birth of live lambs derived from oocytes obtained from donors as young as 4 wk. Defects in the competence of lamb-derived embryos may account for the increased fetal loss during pregnancy and the occurrence of mummified fetuses delivered alongside normal healthy lambs.

Ovum pick-up in sheep: efficiency of in vitro embryo production, vitrification and birth of offspring.

The production of offspring involving available technologies like ovum pick-up, in vitro embryo production and cryopreservation has not been fully described in the sheep. We tested the overall efficiency of these procedures on 20 Sarda dairy ewes that were twice stimulated for recovery of follicular oocytes. In total, 415 oocytes were aspirated from 522 follicles (11.5 oocytes/ewe), and 328 of them (9.1 oocytes/ewe) were selected for in vitro embryo production procedure. Development into blastocysts occurred in 98 embryos (2.7 blastocysts/ewe), of which 64 were vitrified and 34 were transferred, in pairs, directly to recipients. The pregnancy rate, diagnosed at 80 d for fresh and vitrified embryos, did not differ significantly (47.1 vs 42.8%, respectively), but there were significant differences in lambing rates between the 2 groups (41.2 vs 23.8%, respectively). Overall, 24 lambs were born; all weighed within the range for the breed, but head deformities were observed in 2 cases. The results of this study show that with application of the above techniques, it is possible to obtain repeatedly embryos and viable offspring.