Ewe Oocytes Research Articles

Effect of adding growth factors during in vitro maturation on the developmental potentials of ewe oocytes selected by brilliant cresyl blue staining.

Aim:Several factors had been concerned with the developmental competence of the sheep oocyte. This study aims to investigate the effect of adding growth factors (insulin-like growth factor 1 [IGF-1] and epidermal growth factor [EGF]) in the maturation medium of ewe oocytes selected based on brilliant cresyl blue (BCB) screening on in vitro maturation (IVM), fertilization, and pre-implantation embryo development.Materials and Methods:Cumulus-oocyte complexes (COCs) were obtained from the ovaries of slaughtered ewes by either aspiration or slicing techniques. COCs were in vitro matured in a medium containing IGF-1 and EGF (control group). For BCB screening, oocytes were stained and divided into BCB+ oocytes that matured in the same maturation conditions without adding growth factors (Group 2) or in the presence of growth factors (Group 3), and BCB− oocytes that matured in medium without growth factors (Group 4) or with growth factors (Group 5).Results:The supplementation of the maturation medium with growth factors during IVM of (BCB+) oocytes resulted in a significant increase in nuclear maturation rate (90.9%), fertilization rate (75.6%), and embryo developmental rates (60.0%, 46.7%, and 33.3% for cleavage, morula, and blastocyst, respectively).Conclusion:Culturing BCB+ oocytes in a maturation medium containing both EGF and IGF-1 showed a significant improvement in nuclear maturation, fertilization, and pre-implantation embryo development in vitro.

Bisphenol S Impaired In Vitro Ovine Early Developmental Oocyte Competence

Introduction: Bisphenol A (BPA) is a widespread compound in the plastic industry that is especially used to produce baby bottles, food packaging and metal cans. BPA, an endocrine disruptor, leads to alterations in reproductive function and therefore has been banned from the food industry. Unregulated BPA analogues, particularly Bisphenol S (BPS), have emerged and are now used in the plastic industry. Thus, this study aimed to examine the acute effects of low and environmental doses of BPS on ewe oocyte quality and developmental competence, and its mechanism of action, during in vitro maturation. Methods: Ewe cumulus-oocyte complexes underwent in vitro maturation in the presence or absence of BPS (1 nM, 10 nM, 100 nM, 1 µM or 10 µM). Oocytes were then subjected to in vitro fertilisation and development. Results: 1 µM BPS induced a 12.7% decrease in the cleavage rate (p = 0.004) and a 42.6% decrease in the blastocyst rate (p = 0.017) compared to control. The blastocyst rate reduction was also observed with 10 nM BPS. Furthermore, 10 µM BPS reduced the oocyte maturation rate, and 1 µM BPS decreased cumulus cell progesterone secretion. PR and AMH gene expression were reduced in cumulus cells. BPS induced a 5-fold increase in MAPK 3/1 activation (p = 0.04). Conclusions: BPS impaired ewe oocyte developmental competence. The data suggest that BPS might not be a safe BPA analogue. Further studies are required to elucidate its detailed mechanism of action.

Ovine oocytes display a similar germinal vesicle configuration and global DNA methylation at prepubertal and adult ages

Epigenetic mechanisms are thought to be involved in the reduced developmental capacity of early prepubertal ewe oocytes compared to their adult counterparts. In this study, we have analyzed the global DNA methylation pattern and in vitro meiotic and developmental competence of oocytes at the germinal vesicle (GV) stage obtained from adult and 3-month-old donors. All oocytes were aspirated from antral follicles with a diameter ≥3 mm, and DNA methylation on 5-methylcytosine was detected by immunofluorescence using an anti-methyl cytosine antibody. The main global chromatin configuration pattern shown by both prepubertal and adult ovine oocytes corresponded to condensed chromatin localized close to the nuclear envelope (the SNE pattern). Immunofluorescence showed that a global bright nuclear staining of 5-methylcytosine (5-mC) occurred in all germinal vesicle stage oocytes and matched the propidium iodide staining pattern. The total fluorescence intensity values of lamb GVs were not lower than those observed in adult GVs. The meiotic competence and cleavage rates were similar in adult and prepubertal oocytes, however, the developmental competence of embryos to reach blastocysts was higher for adult oocytes than lamb oocytes (p<0.0001). In conclusion, our results indicate that adult-size oocytes derived from 3 to 4 month old prepubertal ewes show similar GV morphology and DNA methylation staining patterns to those obtained from adult animals, despite exhibiting a lower developmental competence.

Low developmental competence and high tolerance to thermal stress of ovine oocytes in the warm compared with the cold season.

Heat stress can potentially affect most aspects of reproduction in mammals. To our knowledge, no studies have ever been conducted for evaluating the influences of hot season on the developmental competence of ewe oocytes. In the present study, for the first time, we evaluated the effects of season (winter or summer), in vitro thermal stress, and their interaction on the ewe oocytes harvested from slaughterhouse ovaries. Cumulus-oocyte complexes (COCs) were either incubated at 39°C for the entire length of IVM period or first incubated at 41°C for 12h and then at 39°C. Evaluated endpoints included the ratios of total aspirated COCs/ovary and good-quality COCs/ovary, the apoptosis (Annexin V staining) and nuclear maturation of oocytes after 24-h IVM, and the developmental competence of oocytes after IVF. Our results showed that the number of aspirated oocytes per ovary was similar in both seasons, but the winter ovaries yielded significantly more oocytes with acceptable morphology in winter than in summer (2.1 ± 0.14 vs. 1.5 ± 0.09, P < 0.05). There was a significant interaction between season and thermal stress on the apoptosis, some nuclear maturation parameters, and blastocyst development of oocytes (P < 0.05). Although the winter oocytes were more developmentally competent than the summer oocytes, the winter oocytes were more sensitive to the thermal stress than summer oocytes. In conclusion, the developmental competence of ovine oocytes was lower in summer than in winter. However, it seemed that summer oocytes were more resistant to the in vitro thermal stress during IVM period compared with winter oocytes.

IMPROVING EWE OOCYTE VIABILITY AFTER VITRIFICATION WARMING USING COMBINATION OF DIFFERENT CRYOPROTECTANT AND CARRIER SYSTEM

The aim of this study was to determine the best combination of cryoprotectant (Ethylene glycol, EG), dimethyl sulfoxide (DMSO) and propanediol (PrOH) and carrier system (hemistraw and cryotop) in improving ewe oocytes viability during cryopreservation. Oocytes with multi layers of compact cumulus cells were colleted from abbatoir and matured in TCM 199 medium supplemented with 10% fetal calf serum for 24-26 h at 38.5° C under 5% CO2 in the air. Matured oocyte was divided into six parts and vitrified in three different vitrification solutions; (i) 17% EG+17% DMSO with hemistraw as carrier system, (ii) 34% EG with hemistraw as carrier system, (iii) 17% EG+17% PrOH in hemistraw (iv), 17% EG+17% DMSO with cryotop as carrier system (v), 34% EG with cryotop as carrier system (vi), and 17% EG+17% PrOH in cryotop. Oocytes were cryopreserved for one week before revived and evaluated for viability. The result showed that oocytes vitrified in media containing EG and DMSO in cryotop had the highest viability (88.16%) compared to media containing EG only or EG and PrOH (70.95% and 68.76%, respectively) (P0.05). Moreover, oocytes viability that vitrified using cryotop and hemistraw as carrier system were not significantly different. The present results indicated that vitrification using combination of EG and DMSO as permeable cryoprotectant and cryotop as carrier system was the best system to maintain oocyte viability after vitrification-warming.

ITRAQ-based proteomic profiling of granulosa cells from lamb and ewe after superstimulation

The number of oocytes obtained from lambs after FSH treatment is far greater than those acquired from adult ewes. However, these oocytes typically have reduced viability in comparison with adult ewe oocytes. However, the molecular mechanisms of differences in viability between lamb and ewe oocytes remain unknown. In the present research, we applied iTRAQ coupled with LC-MS/MS proteomic analysis in order to investigate the proteomic expression profile of granulosa cells from lambs and ewes following stimulation with FSH. We detected 5649 proteins; 574 were differentially expressed between adults and juveniles. Based on Gene Ontology enrichment and KEGG pathway analysis, the majority of DEPs are participated in metabolic processes, ribosome and MAPK signaling pathways. Expression levels in ewes turned out to be lower than lambs. Protein interaction network analysis generated by STRING identified MAPK1, SMAD2, SMAD4, CDK1, FOS and ATM as the major findings among 54 significant differentially expressed of proteins. Quantitative real-time PCR analysis was applied to verify the proteomic analysis. These proteins which were identified in lambs may contribute to the reduction of oocyte quality compared to adults. The present research provides understanding of the molecular mechanism for follicle development in lambs.

Effect of milrinone on the developmental competence of growing lamb oocytes identified with brilliant cresyl blue

Juvenile in vitro embryo transfer is a novel technique that can be used to increase the rate of genetic gain in a population and presents an alternative to embryo technologies on the basis of adult animals. However, oocytes from prepubertal animals have a lower viability than those obtained from adult ewe oocyte donors. In this research, we aimed to determine the optimum concentration and time of treatment of oocytes from prepubertal lambs with brilliant cresyl blue (BCB) stain and milrinone during IVM. This would improve the developmental rate of lamb oocytes and embryos after IVF. First, lamb cumulus–oocyte complexes were cultured under different concentrations (13 or 26 μM) of BCB staining. Treated lamb oocytes were then divided into BCB− (colorless cytoplasm) and BCB+ (colored cytoplasm) groups on the basis of their glucose-6-phosphate dehydrogenase activity. The blastocyst efficiency rate of BCB+ oocytes treated with 13 μM BCB (37.03%) was significantly higher than that of BCB+ oocytes treated with 26 μM BCB (23.25%) and that of nontreated BCB control oocytes (15.37%), as well as that of BCB− oocytes (6.28%). Both control oocytes and BCB+ oocytes exhibited significantly higher cleavage rates (60.15% and 73.44%, respectively) than that of BCB− oocytes (36.19%). Moreover, the diameter and glutathione content of BCB+ oocytes were found to be significantly greater than those of BCB− oocytes (163.37 vs. 159.25 μm and 6.39 vs. 0.26 pM, respectively). After culturing BCB− oocytes in different concentrations of milrinone (0, 50, 75, and 100 μM) for 3, 6, or 9 hours, results reported that supplementation of IVM medium with 75 μM milrinone for 6 hours yielded a significantly higher proportion of blastocysts than the other treatments. These results show that the staining of lamb cumulus–oocyte complexes with 13 μM BCB before IVM may be used to select developmentally competent lamb oocytes. Furthermore, they suggest that milrinone can be used to promote lamb developmental competence of lamb embryos produced during IVF.

Effects of acetyl-L-carnitine on lamb oocyte blastocyst rate, ultrastructure, and mitochondrial DNA copy number

Viable lambs can be produced after transfer of in vitro–derived embryos from oocytes harvested from prepubertal lambs. However, this occurs at a much lower efficiency than from adult ewe oocyte donors. The reduced competence of prepubertal oocytes is believed to be due, at least in part, to deficiencies in cytoplasmic maturation. Differences in the cytoplasmic ultrastructure between prepubertal and adult oocytes have been described in the sheep, pig, and cow. Prepubertal lamb oocytes have been shown to have a different distribution of mitochondria and lipid droplets, and less mitochondria and storage vesicles than their adult counterparts. L-carnitine plays a role in supplying energy to the cell by transporting long-chain fatty acids into mitochondria for β-oxidation to produce ATP. Both L-carnitine and its derivative acetyl-L-carnitine have been reported to increase the blastocyst rate of oocytes from mice, cows, and pigs, treated during IVM. L-carnitine has also been shown to increase mitochondrial biogenesis in adipose cells. Therefore, the aims of this study were to determine if treatment of oocytes from prepubertal lambs with acetyl-L-carnitine during IVM could increase the blastocyst rate and alter mitochondria, vesicle, or lipid droplet number, volume, or distribution. The blastocyst rate was doubled in prepubertal lamb oocytes treated with acetyl-L-carnitine when compared to untreated oocytes (10.0% and 4.6%, respectively; P = 0.028). Light microscopy, scanning electron microscopy, and stereology techniques were used to quantify organelles in untreated and acetyl-L-carnitine–treated lamb oocytes, and quantitative polymerase chain reaction methods were used to measure the mitochondrial DNA copy number. There were no differences in mitochondrial volume, number, or mitochondrial DNA copy number. Acetyl-L-carnitine treatment increased the cytoplasmic volume (P = 0.015) of the oocytes, and there were trends toward an increase in the vesicle volume (P = 0.089) and an altered distribution of lipid droplets (P = 0.076). In conclusion, acetyl-L-carnitine can be used to increase the in vitro blastocyst rate of juvenile oocytes and therefore to improve juvenile in vitro embryo transfer methods. These methods can be used for livestock improvement by increasing the rate of genetic gain. Further work is required to identify the contents of the vesicles and confirm the mode of action of acetyl-L-carnitine in improving oocyte competence.

English

This study was designed to identify the effects of the presence of corpus luteum (CL) on the meiotic competence of the ewe oocytes. In addition, due to the pivotalroles of oocyte recovery methods on efficiency of&nbsp;in vitro&nbsp;maturation and&nbsp;in vitrofertilization, this study considered the new oocyte recovery method &ldquo;Oocyte Recovery with Centrifuge&rdquo; (ORC); Ovine ovaries were obtained from local abattoir at Karaj, Iran. The ovaries were placed in 0.9% saline which was supplemented with penicillin/streptomycin in thermo flask at 37&deg;C and transported to the laboratory within 1 to 2 h of slaughter. The ovaries were assigned to two groups; group-1 with functional CL and group-2 without CL. The oocytes were recovered by means of aspiration pump or ORC. After oocyte recovery, they were cultured in TCM-199 for 24 h. The mean number of oocyte recovery per ovary in group 1 ovaries (1.8 &plusmn; 0.01 via aspiration and 3.84 &plusmn; 0.05 via ORC) was lower (P&lt;0.05) than group 2 ovaries (2.2 &plusmn; 0.00 via aspiration and 5.43 &plusmn; 0.01 via ORC). There were no significant differences (P&gt;0.05) between percentage of nuclear maturation in oocytes which were recovered from group 2 ovaries via aspiration and ORC (75.20 &plusmn; 0.00 vs. 74.95 &plusmn; 0.00, metaphase II; M-II) method. The nuclear maturation in oocytes which were obtained via ORC from group 2 ovaries was higher (P&lt;0.05) than group 1 ovaries (74.95 &plusmn; 0.00 vs.&nbsp;60.07 &plusmn; 0.00b). Nuclear maturation for oocytes obtained via ORC from group 1 ovaries was (P&lt;0.05) lower than oocytes obtained via aspiration (60.07 &plusmn; 0.00 vs. 74.70 &plusmn; 0.00). Result of the present study showed&nbsp;that the presence of CL on ovaries lead to decrease in the quality and the quantity of oocytes. ORC method increased the quantity and quality of recovered oocytes. &nbsp; Key words:&nbsp;Aspiration, corpus luteum,&nbsp;in vitro&nbsp;maturation (IVM), oocyte recovery.

Effect of lanosterol on thein vitromaturation in semi-defined culture system of prepubertal ewe oocytes

The choice of medium and supplements can affect meiotic regulation and may have an impact on the regulation of mammalian oocyte growth and embryonic cell function. The aim of the present study was to assess the effects of oxygen concentration and endogenous lanosterol on the in vitro maturation (IVM) media without serum and based on recombinant human chorionic gonadotrophin in prepubertal ewe oocytes. Firstly, the effect of varying oxygen concentrations (5% and 20%) during IVM in TCM-199 supplemented (4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM sodium pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH; Gonal-F® 75 UI, Serono, Italy), 0.1 UI/ml recombinant leuteinizing hormone (r-LH; Lhadi® 75 UI, Serono, Italy) and 1 μg/ml estradiol-17β) on subsequent nuclear maturation of oocytes examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) was investigated. Secondly, two concentrations of lanosterol (0, 10 and 50 μM) were added to the IVM medium. Nuclear maturation of oocytes was examined as previously. Lipid content in oocytes, an important indicator of cytoplasmic maturity, was also measured using Nile red fluorescent stain. The results showed that low oxygen concentration affected the nuclear maturation. Similarly, a significantly higher rate of meiosis resumption was observed with 10 μM (72.3%) of lanosterol compared with the control (51.8%) or 50 μM of lanosterol (59.4%). A significantly higher content of lipids was also observed with 10 and 50 μM of lanosterol (7.3 ± 0.2 × 10(6) and 7.4 ± 0.2 × 10(6) arbitrary units of fluorescence) compared with the control (6.7 ± 0.2 × 10(6) arbitrary units of fluorescence). The results indicate that 10 μM lanosterol during IVM in medium without serum and based on recombinant human chorionic gonadotrophin has a positive effect on maturation of prepubertal ewe oocytes.

334 EFFECT OF A GROWTH MEDIUM DURING IVM ON EMBRYO DEVELOPMENT OF PREPUBERTAL EWE OOCYTES

The aim of this study was to assess the effect of an in vitro growth medium (De Wu et al. 2006 Biol. Rep. 75, 547-554) in prepubertal ewe oocytes selected by the brilliant cresyl blue (BCB) test. Prepubertal ewe oocytes were recovered by slicing ovaries of slaughtered animals and immediately exposed during 1 h to 13 μM BCB and classified according to their cytoplasm coloration (Rodriguez-Gonzalez E et al. 2002 Theri- ogenology 57(5), 1397-1409): BCB+ (blue cytoplasm, hypothetically grown oocytes) and BCB- (uncolored cytoplasm, hypothetically growing oocytes). Uncolored oocytes (BCB-) were matured using three culture media: growth medium (GM: TCM-199, 0.04 μg mL-1 FSH, 0.04 μg mL-1 LH, 0.004 μg mL-1 estradiol, 100 μg mL-1 ascorbic acid, and 5 μL mL-1 ITS: insulin transferrin selenium), conventional maturation medium (CM: TCM-199, 10 μg mL-1 FSH, 10 μg mL-1 LH and 1 μg mL-1 estradiol) and modified maturation medium (MM: CM with the addition of 100 μg mL-1 ascorbic acid and 5 μL mL-1 ITS). Oocytes were matured in GM for 12 h and then separated into 2 groups, CM (GM+CM) and MM (GM+MM) for another 12 h of maturation. Two extra groups of BCB- oocytes were directly cultured for 24 h in CM or MM media (BCB-/CM and BCB-/MM). Colored oocytes (BCB+) and a control group (oocytes not exposed to BCB) were matured for 24 h in CM. All groups were cultured at 38.5°C and 5% CO2 in humidified atmosphere. Fertilization took place in SOF medium supplemented with 10% of estrous sheep serum during 20 h with a sperm concentration of 1 × 106 spermatozoa/mL. Presumptive zygotes were cultured for 8 days in SOF with 10% FCS at 38.5°C, 5% CO2 and 5% O2. Results are shown in Table 1. The percentage of morula plus blastocyst obtained from BCB - oocytes was significantly increased in oocytes exposed to growth medium (containing ITS, ascorbic acid and low hormone concentrations; groups GM+CM and GM+MM) for the first 12 h. An increasing tendency has also been observed in blastocyst yield in these two groups. Regarding maturation rate, no differences were found in all groups (data not shown). In conclusion, as De Wu et al. (2006) showed in prepubertal gilts, we also achieved some improvements in embryo development of growing oocytes when the first 12 h of maturation took place in a growth medium. However, embryo developmental potential of BCB- oocytes is still lower compared with that of BCB+ oocytes. Table 1.Effect of GM on embryo development of BCB- oocytes Grant sponsor Spanish Ministry of Science and Innovation.Code: AGL2007-60227-CO2-01

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 °C and 15 °C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 °C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 °C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 μL semen at a sperm concentration of 20 × 10 6/mL. Semen was stored at 5 °C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 °C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 °C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly ( P < 0.001) up to 72 h after semen collection.

Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen

No laboratory test exists that can reliably predict differences among rams in field fertility after artificial insemination (AI) with frozen-thawed semen. In vitro fertilisation (IVF) has been proposed as a method of predicting these differences. The objectives of this study were to evaluate whether IVF system could discriminate among rams of different fertility in vivo after AI using frozen-thawed semen. Also, to examine effects of lowering sperm concentration on discrimination power between rams used for IVF. The aim of Experiment 1 was to evaluate the effect of altering the sperm concentration from 2 × 10 6 to 0.03125 × 10 6 spermatozoa/mL on subsequent cleavage rate and blastocyst rate in vitro. In Experiment 2, six rams (three High and three Low in vivo fertility; average pregnancy rates of 37.6% and 21.8%, respectively) were compared for their fertilising ability in IVF. Spermatozoa from each of the six rams were added to ewe oocytes using a concentration of either 2 × 10 6 or 0.0625 × 10 6/mL. There were six replicates with 25 oocytes per well and two wells per ram per replicate. Cleavage rate was monitored at 48 h post-insemination (p.i.) and blastocyst rate determined on Days 6–8 p.i. In Experiment 1, cleavage rate increased with increasing sperm concentration and blastocyst rate was not affected by sperm concentration on any day. When the six rams were tested using 2 × 10 6 spermatozoa/mL, no significant differences were found between High and Low fertility groups for cleavage rate or blastocyst rate on Days 6, 7, or 8 p.i. ( P > 0.05). When the experiment was repeated using 0.0625 × 10 6 spermatozoa/mL, no differences were found between High and Low group rams for blastocyst rate on any of Days 6, 7 or 8 p.i. ( P > 0.05). However, there was a significant difference between High and Low fertility rams for percentage of oocytes cleaved (16.4, S.E. 2.02%; P < 0.01) and the correlation between fertility in vivo and cleavage rate in vitro was significant ( P = 0.013). Replicate of IVF was a source of significant variation for both cleavage rate and blastocyst rate and conditions need to be further controlled. However, we suggest that using a low concentration of spermatozoa (0.0625 × 10 6/mL) for IVF may be a useful method for predicting field fertility of frozen-thawed ram semen.

In vitro and in vivo developmental capabilities and kinetics of in vitro development of in vitro matured oocytes from adult, unstimulated and hormone-stimulated prepubertal ewes

The in vitro and in vivo developmental capabilities and kinetics of in vitro development of embryos derived from adult ewes and from unstimulated (16- to 24-week-old) and hormone-stimulated prepubertal (3- to 5-week-old) ewes were assessed. Cleavage was lower for hormone-stimulated (617/1025; 60.2%) than unstimulated prepubertal (117/169; 69.2%) and adult ewe oocytes (184/267; 68.9%; P < 0.05). Blastocyst formation by Day 7 (from zygotes) was similar for unstimulated (45/117; 38.5%), hormone-stimulated prepubertal (229/617; 37.1%) and adult ewes (101/184; 54.9%). Blastocysts derived from hormone-stimulated prepubertal ewes developed mainly on day 7, compared with Day 6 for adult and unstimulated prepubertal ewes. Pregnancy rates (day 60) and embryonic loss (between Days 20 and 60) did not differ after transfer to adult recipient ewes of adult, unstimulated and hormone-stimulated prepubertal-derived fresh or frozen-thawed embryos. The number of lambs born as a proportion of embryos transferred did not differ for fresh and frozen embryos derived from adult ewes (3/16; 18.8% and 1/12; 8.3%, respectively) and unstimulated prepubertal lambs (2/6; 33.3%, and 1/10; 10.0%, respectively), but was higher for fresh than frozen embryos from hormone-stimulated prepubertal ewes (7/16; 43.8%, and 2/14; 14.3%, respectively; P < 0.05). There were high rates of in vitro and in vivo development of oocytes from 3- to 5-week-old lambs, but in vitro development was lower than with oocytes from adult ewes. However, the speed of embryonic development in vitro and the in vivo development of fresh and frozen embryos were similar to those derived from adult and unstimulated prepubertal ewes. The present results were an improvement in the efficiency of producing embryos and offspring from hormone-stimulated 3- to 5-week-old lambs.

The effect of gamete co-incubation time during in vitro fertilization with frozen-thawed unsorted and sex-sorted ram spermatozoa on the development of in vitro matured adult and prepubertal ewe oocytes

In vitro matured adult (Experiment 1) and prepubertal (Experiment 2) ewe oocytes were co-incubated with unsorted or sex-sorted frozen-thawed spermatozoa for 2 to 3 h (short) or 18 to 20 h (long) to determine the effects of reducing the gamete co-incubation time during IVF on subsequent embryonic development in vitro. For oocytes derived from adult ewes, there were no differences in oocyte fertilization and cleavage at 24 h post insemination (hpi) between types of spermatozoa or co-incubation times ( P > 0.05). By 48 hpi, oocyte cleavage was higher after a short (390/602, 64.8%) compared with a long (381/617, 61.7%) co-incubation ( P < 0.05), and was not significantly different for unsorted (266/372, 71.5%) and sex-sorted (505/849, 59.9%) spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (150/266, 56.4%) and sex-sorted (295/505, 58.4%) spermatozoa, but was higher after a short (240/390, 61.5%) than long (205/381, 53.8%) co-incubation ( P < 0.05). Oocyte development to the blastocyst stage was not different for unsorted (150/372; 40.3%) and sex-sorted (295/847; 34.8%) spermatozoa but was significantly increased by a short (240/602, 39.9%) compared with a long (205/617, 33.2%) co-incubation. Fertilization of oocytes from prepubertal ewes was similar for types of spermatozoa and for duration of co-incubation. Oocyte cleavage (48 hpi) was similar for a short (241/377, 63.9%) and long (226/349, 64.8%) co-incubation with unsorted spermatozoa, but was increased ( P < 0.05) by a long co-incubation (286/500, 57.2% versus 163/517, 31.5%) with sex-sorted spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (230/467, 49.3%) and sex-sorted (186/449, 41.4%) spermatozoa, and a short (200/404, 49.5%) or long (216/512, 42.1%) co-incubation. However, oocyte development to the blastocyst stage was higher ( P < 0.05) after IVF with unsorted (230/726, 37.1%) than sex-sorted (186/1017, 18.3%) spermatozoa. Reducing the duration of gamete co-incubation did not deleteriously affect the in vitro development of adult and prepubertal ewe derived oocytes after IVF with unsorted and sex-sorted spermatozoa. In general, sex-sorting had no substantial influence on fertilization and embryo development rates.

The Effect of sheep serum and estrus sheep serum on in vitro maturation and fertility rate of ewe oocyte

The main objective of this research was to obtain the effect of sheep serum (SS) and estrus sheep serum (ESS) on in vitro maturation oocyte and ovine fertilization. This study was carried out in experimental laboratory in animal reproduction laboratory, Faculty of Animal Husbandry, Padjadjaran University. Results showed that treatments significantly (P 0.05) results observed on ovine in vitro fertilization (1, 2 and >2 pronuclei). Concentration of 10-20% ESS in CR1aa media were significantly (P<0,05) better than that of SS on maturation rate of ovine oocyte. Key Words: Sheep Serum, Estrus Sheep Serum, Maturation, Fertilization

A comparison of two recovery methods of ovine oocytes for in vitro maturation

Ewe and ewe lamb ovaries obtained from a slaughterhouse were used to study the influence of the oocyte collection technique (aspiration and slicing) on the quantity and quality of immature oocytes recovered during the non-breeding season. In the aspiration technique, all visible follicles (2–6 mm in diameters) were aspirated with a 2 ml syringe (20-gauge needle) using PBS containing heparin and in the slicing technique; the follicles were incised with the aid of scalpel blade. The oocytes were classified on the basis of the cumulus cover and cytoplasmic appearance into four classes. The total number of ewe oocytes recovered and the yield of good-quality oocytes recovered per ovary by aspiration and slicing were 8.1 and 1.8, and 6.3 and 3.3, respectively. The corresponding values for ewe lambs were 3.7 and 0.8, and 4 and 1.7, respectively. The recovery of good quality oocytes using the slicing technique yielded more oocytes per ovary than aspiration in both ewe and ewe lambs during the non-breeding season.

In vivo and in vitro fertilizing capacity of cryopreserved European mouflon [ Ovis gmelini musimon] spermatozoa used to restore genetically rare and isolated populations

European mouflon sheep are an endangered species of ovidae residing primarily in the mountenous habitat of the islands of Sardinia and Corsica. The purpose of this study was to assess the fertilizing capacity of cryopreserved European mouflon spermatozoa after AI in synchronized mouflon and domestic ewes and after IVF in in vitro matured mouflon and domestic ewe oocytes collected by OPU technique. Domestic ram ( Ovis aries) spermatozoa served as control. Semen was collected by artificial vagina from three mouflons and three domestic rams during the breeding season and was cryopreserved. At thawing, no significant differences in sperm viability were found between the wild and the domestic species (53.1 ± 4.6% versus 56.0 ± 4.7%) whereas the percentage of acrosome-intact sperm was lower in mouflon (55.5 ± 4.6%) than in ram semen (62.7 ± 3.1%; P < 0.05). Lambing rate did not differ between synchronized mouflon and domestic ewes (5/11 versus 8/12) after 150 and 156 days of pregnancy, respectively. After two OPU sessions, 87 and 132 oocytes were collected from three hyperstimulated mouflon and three domestic ewes. Cryopreserved/thawed semen was inseminated with an endoscope into the uterus of corresponding species during the non-breeding season. The oocytes were matured and fertilized in vitro; 61/73 mouflon and 81/101 domestic ewe oocytes were found to be fertilized. From these, we obtained 6/61 and 17/81 blastocysts. After vitrification and thawing, the hatching rate showed no significant difference between mouflon and sheep blastocysts (4/6 versus 14/17). In conclusion, our data showed that cryopreserved mouflon spermatozoa can be successfully used to carry out a genuine and complete program of genetic restoration in small and isolated groups of European mouflons.

205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.

Maturation status, protein synthesis and developmental competence of oocytes derived from lambs and ewes.

The efficacy of oocyte selection for in vitro embryo production depends on the abundance and diameter of follicles, cumulus layers around the oocytes and subsequent fertilization. Application of 'ovum pick-up' technique allows us to utilize partially matured oocytes for embryo production even from juvenile subjects. To compare their developmental competence, oocytes derived from lambs and ewes and cultured in maturation medium for up to 26 h were assessed at 2 h intervals by confocal microscopy after chromatin and microtubulin-specific fluorochrome labelling. Lamb oocytes reached second meiotic metaphase (MII) at lower numbers at 24 h (60.0%) and 26 h (28.6%) whereas 85.7% of adult-derived oocytes attained MII status by 24 h of maturation. Radiolabelling of oocyte proteins revealed higher incorporation of [(35)S-]-methionine and [(35)S]-cysteine in adult-derived oocytes compared to lamb oocytes. Although the cleavage rate of lamb oocytes was similar to that of ewe oocytes, the proportion reaching blastocyst stage was significantly lower (p < 0.05) in the lamb-derived oocytes. However, blastocysts from both types of oocytes displayed similar cell lineage allocations to inner cell mass and trophectoderm.