Biomedical subjects

S Naitana

Publications and source records attributed to S Naitana.

At least 19 recordsLinked to original sources

PubMed2007

Relations between relative mRNA abundance and developmental competence of ovine oocytes.

The present study was conducted to investigate the relation between in vitro developmental competence and the expression of a panel of developmentally important genes in germinal vesicle (GV) stage oocytes. One-month-old prepubertal and adult sheep oocytes were used as models of low and high quality gametes, respectively. Cumulus-oocyte complexes (COCs) derived from lambs and ewes were in vitro matured and fertilized, and their cleavage rate at 22, 26, and 32 hr post fertilization and the blastocyst yield were observed to assess their developmental potential. In parallel, the relative abundance (RA) of 11 genes was analyzed by semi-quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) assay in the two groups of oocytes. We observed similar maturation and fertilization rates in the two groups, but a significant lower rate of cleaved prepubertal oocytes (P < 0.05), a general delay in the timing of their first division (P < 0.01), and a lower blastocysts production (P < 0.05). The analysis of gene expression evidenced no difference in the RA of four transcripts [superoxide dismutase (SOD), ubiquitin, beta-actin, cyclin B] in the two classes of oocytes, but a statistically lower RA of seven messenger RNAs (mRNA) [Na(+)K(+)ATPase, p34(cdc2), Glucose-transporter I (Glut-1), Activin, Zona Occludens Protein 2 (PanZO2), Poli(A)Polymerase (PAP), E-Cadherin (E-Cad)] in the prepubertal oocytes compared to the adult ones. The present data show for the first time in the ovine species that the lower developmental competence is associated with deficiencies in the mRNAs storage during the oocyte growth.

Aging↗

PubMed2006

Association between numbers of ovarian follicles in the first follicle wave and superovulatory response in ewes.

The aim of this study was to examine the variability in the number of ovarian follicles in sheep and to determine if the average number of follicles per day influences the response to superovulation and resulting embryo quality. Ewes (n=83) were synchronized and the number of follicles (> or =2 mm diameter) in the ovaries were counted daily between Days 0 and 4 of the oestrous cycle using transrectal ultrasonography. Fourteen to 21 days later, 47 ewes were randomly chosen from the group and were treated with an intravaginal progestagen pessary for 12 days and superovulated with 1500 IU eCG administered as a single injection 10 days after sponge insertion. Ewes were mated and reproductive tracts were recovered after slaughter on Day 6 of pregnancy. The number of corpora lutea was counted, uterine horns were flushed and the morphology and developmental stage of the recovered oocytes/embryos was assessed. The mean daily number (+/-S.D.) (> or =2 mm diameter) of follicles per ewe was 8.5+/-2.8 (ranging between 3 and 16). After superovulation animals with few follicles (Low group: <8 follicles/day; n=21) had fewer (P<0.005) corpora lutea, total structures (unfertilized oocytes and embryos), good quality and total embryos compared to animals with many follicles (High group: > or =8 follicles/day; n=23). No difference was found in the proportion of good quality embryos (relative to the total number; Low 0.68+/-0.11 versus High 0.79+/-0.08; P=0.21) between the two groups, or the recovery rate, the number of unfertilized oocytes or the number of poor quality embryos per animal. We conclude that ewes with a higher number of follicles (> or =8) during the first follicular wave had a better superovulatory response (in terms of corpora lutea and high quality embryos) 2-3 weeks later; however, there was no relationship between the number of follicles and the proportion of good quality embryos per animal.

Animals↗

PubMed2006

Effects of progestagens on follicular growth and oocyte developmental competence in FSH-treated ewes.

Previous research has reported evidence for negative effects of progestagens on follicular growth and oocyte competence. In the present study, negative effects of progestagens on follicular growth and oocyte developmental competence were assessed. During the breeding season, 20 Sarda ewes were treated with two doses of cloprostenol, 10 days apart, to assure the presence of a corpus luteum (CL). On day 5 after the second cloprostenol dose, 10 ewes were treated with a progestagen sponge while 10 females remained untreated. Starting on day 7 after the second cloprostenol dose, all the ewes were treated with 6 equal doses of 24 I.U. of FSH (Ovagen, ICP, NZ), every 12h. The number of follicles > or =2mm in diameter increased (P<0.0005) in all the ewes from 24 h before to 60 h after the first FSH dose (from 12.8+/-1.1 to 23.4+/-1.3 in treated and from 12+/-0.6 to 22+/-1.2 in untreated ewes, n.s.). There were no significant differences in follicle dynamics between groups, but concentrations of estradiol in control ewes were higher than in the progestagen group (P<0.05). Twelve hours after the last FSH dose, oocytes were collected by ovum pick-up. Recovery rates were lower for progestagen-treated ewes (71.1 versus 83%; P<0.001). After IVP procedure, cleavage rate was also lower in the progestagen group (39.1 versus 82.6%; P<0.001). Furthermore, blastocysts output revealed that oocyte developmental competence was lower in progestagen group (17.3 versus 30.4%; P=0.245), although differences were not significant. These results suggest deleterious effects from progestagen on oocyte developmental competence and set the basis for new protocols for in vitro embryo production.

Animals↗

PubMed2005

GnRH antagonist enhance follicular growth in FSH-treated sheep but affect developmental competence of oocytes collected by ovum pick-up.

This study evaluates the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from sheep treated with GnRH antagonists (GnRHa) and high doses of FSH. Eighteen Sarda ewes were treated with progestagen sponges (day 0). On day 7, 10 ewes received 3 mg of GnRHa s.c., while 8 served as control receiving saline. On day 10, all animals were treated with 96 IU of ovine FSH in four equal doses given i.m. every 12 h. We monitored follicular development by ultrasonography, twice daily from day 7 to 11, and found that GnRHa induced a significant increase in the number of total follicles in 72 h (11.7+/-0.9 to 21+/-2.4, r(2)=0.598, P<0.0001), while this number remained stable in control sheep. We found that FSH induced a significant rise in the number of follicles in both groups; but always higher (P<0.05) in GnRHa treated sheep, confirming that GnRHa enhances ovarian response to exogenous FSH stimulation. Twelve hours after the last FSH dose, oocytes were collected by OPU. Recovery percentage, morphological quality, ability to resume meiosis, fertilization and cleavage were similar in oocytes from treated and untreated sheep. However, the final blastocysts output was lower in GnRHa group (10.1% versus 27.4% in control group; P<0.05). In addition, re-expansion rates after vitrification, thawing and in vitro culture were lower in GnRHa treated ewes, although differences did not reach statistical significance (55.5% versus 74.1% in GnRHa treated and in control sheep, respectively).

Animals↗

PubMed2005

Delay on the in vitro kinetic development of prepubertal ovine embryos.

In the present study we characterize the developmental potential of prepubertal and adult ovine oocytes, analyzing the developmental speed to two-cell and blastocyst stages and its relationship with hatching from the zona pellucida, development after vitrification and the number and allocation of inner mass and trophoblastic cells. Prepubertal and adult ovine oocytes were matured and fertilized in vitro and first cleavage rates at 22, 26 and 32 h were recorded. Cleaved oocytes were cultured and blastocyst production was assessed at 6-9 days post-fertilization (dpf). Blastocysts from the two sources obtained on different days were divided into two groups: the first was vitrified, warmed and cultured in vitro to evaluate re-expansion of the blastocoelic cavity; blastocysts of the second were cultured separately to allow for hatching and count of trophoblastic and inner mass cells of hatched blastocysts by differential staining. We observed a significantly lower rate (P < 0.01) of cleaved prepubertal oocytes at 22 and 26 h after fertilization while it was higher (P<0.01) at 32 h than in the adult ones. Adult blastocyst production was significantly lower (P < 0.01) in prepubertal than in adult groups and began on the seventh dpf, later (P < 0.01) than in the adult group, where they appeared on the sixth dpf. Prepubertal blastocysts hatched at a lower rate than the adult ones (P < 0.01) and in both experimental groups faster blastocysts showed a higher (P < 0.01) hatching rate. Similarly, prepubertal derived blastocysts showed lower viability after vitrification (P < 0.01) compared to the adult counterparts, and in particular slower embryos had reduced viability after vitrification compared to the fastest (P < 0.01). Cell number was not different between blastocysts of both groups obtained at 6 and 7 dpf, which were higher (P < 0.01) than those obtained at 8 and 9 dpf. The ICM/trophoblast cell ratio was similar in 6- and 7-day obtained blastocyst and increased (P < 0.01) in those obtained 1 or 2 days later. These findings show that differences in kinetic development between prepubertal and adult derived embryos reflect differences in developmental capacity of the oocytes from which they derive and could be indicative of embryo quality.

Animals↗

PubMed2005

Induction of the presence of corpus luteum during superovulatory treatments enhances in vivo and in vitro blastocysts output in sheep.

This report offers the results of two experiments developed to test possible benefitial effects of the presence of corpus luteum (CL) on in vivo and in vitro sheep embryo production; using two different breeds treated with two different protocols by two different teams at two different centres. In the first trial, estrus was synchronized in 11 ewes with two doses of cloprostenol, 10 days apart. On day 1 after estimated ovulation, sheep were treated with progestagen sponges and superovulated with eight decreasing doses (26.4 units NIH-FSH-S1 x 3, 22.0 units x 2, and 17.6 units x 3) of ovine FSH injected twice daily. Ovulation rate and number of embryos obtained in vivo were compared to those from 12 control ewes without cloprostenol treatment. Presence of a CL improves the number of transferable embryos (7.4+/-0.6 versus 4.1+/-0.6 in control ewes, P < 0.05). The second trial investigated the effects of the presence of CL on embryos produced in vitro from six ewes bearing CL and six ewes without CL at start a superovulatory treatment consisting of 96 units of ovine FSH administered in four equal doses given every 12 h. There were not detected effects of the CL on the number and size of follicles or in the number, morphology and ability to resume meiosis of their oocytes. However, oocytes from ewes with CL showed higher rates of fertilization (73.5 versus 45.5%, P < 0.005), higher development to blastocyst (35.8 versus 19.3%, P < 0.01) and higher hatching rates after vitrification (80.0 versus 25.0%, P < 0.05).

Animals↗

PubMed2004

FSH different regimes affect the developmental capacity and cryotolerance of embryos derived from oocytes collected by ovum pick-up in donor sheep.

The objective of the present study was to compare the developmental capacity of sheep oocytes obtained by OPU after two different ovarian stimulations, and cryotolerance to vitrification procedures of in vitro derived embryos after in vitro maturation, fertilisation and culture of these oocytes. Sheep were divided into three groups: (A) no treatment (control); (B) constant doses of FSH (FSH-c); (C) decreasing doses of FSH (FSH-d). Ovine groups FSH-c and FSH-d were synchronised by the insertion of intravaginal sponges left in situ for 7 days; FSH (total dose: 96IU) was administered in four doses given every 12h starting on Day 5. Twelve hours after the last FSH administration oocytes were collected by OPU technique. The control group showed a significantly lower number ( P<0.05 ) of follicles (166) than FSH-c (294) and FSH-d (317) groups, while the number of follicles >5mm was significantly higher ( P<0.01 ) in FSH-d group, showing that this protocol stimulates the growth of a different follicle population compared to FSH-c group. The control group showed a higher number of <2mm follicles ( P<0.01 ). We did not find any difference in oocyte quality between the three groups and therefore the percentage of discarded oocytes was similar. No significant differences were found between control, FSH-c and FSH-d groups in terms of maturation (90.9, 85.7 and 87.7%, respectively) and fertilisation rates (75.2, 80.9 and 83.7%, respectively) while a significantly higher ( P<0.01 ) blastocyst rate was observed in the FSH-c group than in the FSH-d and control groups (20.4% versus 11.8 and 13.7%, respectively). After vitrification, warming and 72 h in vitro culture, the hatching rate was significantly higher ( P<0.01 ) in the control (87.5%) and FSH-c (90.5%) groups than in the FSH-d group (66.7%). Control and FSH-c groups showed a significantly higher ( P<0.001 ) number of total cells than FSH-d group ( 217.6+/-26.5 and 203.0+/-33.2 versus 147.5+/-20.2 ), while no differences were observed in ICM cell rates in the control ( 35.6+/-3.8 ), FSH-c ( 37.1+/-4.6 ) and FSH-d ( 36.6+/-6.7 ) groups. These results indicate that donor sheep stimulated with FSH-c produced better quality oocytes and blastocysts showing better cryotolerance than ewes given the decreasing doses treatment.

Animals↗

PubMed2004

M-phase promoting factor (MPF) and mitogen activated protein kinases (MAPK) activities of domestic cat oocytes matured in vitro and in vivo.

This work was undertaken in order to examine M-phase promoting factor (MPF) and mitogen-activated protein kinases (MAPK) activities during meiotic progression of cat oocytes cultured in two different media for two different incubation times and preovulatory cat oocytes that reached MII in vivo. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 or SOF for 24 h and 40 h. In vivo matured oocytes were recovered by follicular aspiration from ovaries of domestic cats ovariectomized 24 h to 26 h after hormonal treatment. Results showed that the kinetic of MPF and MAPK activity was similar during meiotic progression of cat oocytes matured in TCM 199 and SOF. After 24 h of incubation, MII oocytes had significantly (p < 0.001) higher MPF and MAPK levels than MII oocytes cultured for 40 h in both culture media. MPF and MAPK activity was significantly (p < 0.01) lower in the oocytes matured in vitro than in those matured in vivo. This study provides evidence that the two different maturation media did not determine differences in MPF and MAPK fluctuations and levels during meiotic progression of cat oocytes and that the time of maturation influenced the level of the two kinases. Moreover, it shows that MPF and MPK activity is higher in in vivo matured oocytes than in in vitro matured oocytes, suggesting a possible incomplete cytoplasmic maturation after culture.

Animals↗

PubMed2002

The effect of co-culture on the development of in vitro matured equine oocytes after intracytoplastic sperm injection.

It is clear that, in the horse, there are many weak links in the process of in vitro embryo production; an optimal culture system for equine oocytes does not exist, and related data are conflicting. Therefore, the ability of 3 different culture systems to support embryonic development of ICSI horse oocytes was examined. Oocytes (n = 261) suitable for culture were collected from 55 ovaries and divided, according to cumulus morphology, into 2 categories: expanded cumulus and compacted cumulus. Oocytes with expanded and compacted cumulus were cultured for in vitro maturation in TCM 199 + 10% FCS + 0.1 iu/ml FSH/LH at 38.5 degrees C under 5% CO2 in air for 24 and 40 h, respectively. Oocytes (n = 149) reached metaphase II and were subjected to ICSI with frozen semen and then incubated in 3 different culture systems: A) TCM 199 + 10% FCS alone or B) on granulosa cell monolayer, C) SOF + MEM amino acids + 0.8% BSA. Cultural conditions were 39 degrees C and 5% CO2 in air for A and B, while a gas mixture (5% CO2, 5% O2, 90% N2) was used for C. The fertilisation rate was 32%. The cleavage rate in Group A was 74.4% (32/43); 18 embryos reached 2-6 cell stage, eight 8-16 cell, four 16-32 cell and two >32 cell. In Group B, the cleavage rate was 73.5% (36/49) with better results in embryonic development; 14 reached 2-6 cell stage, eighteen 8-16 cell, twelve 16-32 cell and five >32 cell. In Group C, the cleavage rate was significantly lower then in A and B; only 15 of 47 ICSI oocytes (39.1%) cleaved with maximum development to 2-6 cell stage. The remaining oocytes (68.1%) degenerated during culture. In conclusion, IVM horse oocytes can be fertilised in vitro with high efficiency with ICSI and co-culture systems showed to be superior in supporting in vitro embryo culture compared to simple ones. The identification of the factors beneficial to in vitro embryo development provided by the somatic cells could be important to optimise the embryo culture systems for equine embryos.

Animals↗

PubMed2001

Intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa.

The ability to mature and fertilize oocytes of endangered species may allow us to sustain genetic and global biodiversity. The first objective of this study was to compare the effect of two different culture media and two different incubation times on in vitro maturation (IVM) of domestic cat oocytes. The second objective was to determine the developmental competence of in vitro matured cat oocytes after intracytoplasmic sperm injection (ICSI) with cat spermatozoa. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 medium or in synthetic oviductal fluid (SOF), both of which were supplemented with cysteamine, BSA, FSH, LH. Nuclear maturation was assessed after 24 h and 40 h of incubation. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 40 h of incubation were significantly higher (P<0.001) after culture with SOF (88/110, 80% and 159/192, 82.8%) than TCM 199 (86/129, 66.7% and 58/90, 64.4%). Oocytes (n = 231) matured in vitro in SOF for 24 h were fertilized by ICSI with frozen-thawed epididymal cat spermatozoa. After ICSI, one group of oocytes (n = 129) was activated with ethanol, and a second group (n = 102) was not activated. The developmental competence of all ICSI oocytes was examined after 7 days of in vitro culture. After 28 h of culture, the cleavage frequency of ICSI-activated oocytes was significantly higher (P<0.001) than that of IC

Animals↗

PubMed2001

Oocyte cryopreservation and ovarian tissue banking.

Oocyte cryopreservation, despite its impact on conservation of genetic resources, is not yet an established technology. Several problems need to be solved before this technology can be applied regularly. Chilling membrane susceptibility and formation of ice due to the large volume of the cell are the major problems observed. However, during the last years, several studies were done to obtain viable oocytes after cryopreservation. The addition of molecules known to stabilize membranes and the creation of freezing systems with rapid cooling throughout the transition phase have yielded a good percentage of viable immature and mature oocytes More recently, storage of female gametes was achieved by cryopreservation of cortical ovarian tissue. The possibility of restoring fertility by transplantation of frozen ovarian tissue or its long-term culture in vitro represents an important future means of preserving the fertility of patients and of storing the gametes of rare animals.

Animals↗

PubMed2001

Cell coupling and maturation-promoting factor activity in in vitro-matured prepubertal and adult sheep oocytes.

We examined some differences between prepubertal and adult ovine oocytes; in particular we analyzed the functional status of the cumulus-oocyte complex, protein synthesis during in vitro maturation, and because no information is available on prepubertal and adult sheep, maturation-promoting factor (MPF) fluctuations throughout meiotic progression both in prepubertal and adult sheep oocytes. After 24 h of maturation, percentages of MII oocytes were similar between prepubertal and adult animals. Electron microscopy examinations showed that prepubertal oocytes had fewer transzonal projections than adult oocytes. Methionine uptake was significantly lower in prepubertal cumulus-enclosed oocytes examined through meiotic progression. On the contrary, denuded prepubertal oocytes showed a higher methionine incorporation in the first 4 h of incubation compared with adult oocytes. We also found some differences in MPF activity between prepubertal and adult oocytes at MII stage. In fact, prepubertal MII oocytes had a significantly lower level of MPF activity than adult oocytes did and, after fusion with germinal vesicle oocytes, they were unable to induce nuclear breakdown and chromosome condensation 1-2 h post-fusion, whereas adult MII oocytes could induce these processes. Our findings show that the lesser competence of prepubertal oocytes could be due to morphological anomalies and alterations in physiological activity and that oocytes do not reach full developmental competence until puberty.

Animals↗

PubMed2000

Vasoactive intestinal peptide influences hatching of ovine blastocysts.

Expanded blastocysts collected from superovulated Sarda ewes were divided at random into four groups for culture in a simple medium that does not support blastocyst hatching (CZB) or a complex medium that is permissive to hatching (TCM 199), with or without vasoactive intestinal peptide (VIP), a known embryo mitogenic peptide. Plasminogen activator (PA) secretion after 24 h of culture, and the number of cells, diameter of blastocysts and hatching rate after 48 h of culture were compared. The results showed an increase in hatching rate (78.6 v. 6.7%; P<0.01), diameter and number of cells (220.89 v. 210.44 microm, P<0.01 and 246 v. 232, P<0.01 respectively) and caseinolytic areas (1.33 v. 0.92 cm, P<0.01) of blastocysts cultured in TCM 199 compared with those cultured in CZB. Supplementation of the culture media with VIP increased these parameters in CZB (P<0.01) and partially in TCM 199. In particular, cell number, diameter and PA activity were significantly higher (P<0.01) after culture with VIP in both media. Immunoneutralization of exogenous VIP in culture with anti-VIP antibody caused a decrease in the hatching rate (P<0.01) of embryos cultured in medium with VIP, similar to the rate in unsupplemented CZB (P<0.01). These results suggest a receptor-mediated response. In immunohistochemical studies, VIP was shown to bind receptors in hatched blastocysts demonstrating the VIP-receptor interaction, and VIP receptors of approximately 150 kDa were revealed by electrophoretic studies. In conclusion, ovine preimplantation embryos exhibit VIP receptors, providing a basis for a receptor-mediated influence of VIP in the hatching of ovine blastocysts.

Animals↗

PubMed2000

Activity of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) after parthenogenetic activation of ovine oocytes.

The maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are the key regulators of both meiotic and mitotic cell cycles. Knowledge of the dynamics of these two kinases during the transition from meiosis to mitosis would be of great importance for cloning by nuclear transfer. In this study, experiments were designed to assay the changes of MPF and MAP kinase activity of in vitro matured ovine oocytes after chemical activation and culture in 0 mM or 2 mM 6-dimethylaminopurine (6-DMAP) for 12 h. Moreover, to determine the biological significance of the fluctuations of MPF, activated oocytes were fused with GV-staged partners. The biochemical results showed that the high MPF activity of MII oocytes fell to basal level precipitously within the first hour after activation, started to increase at 6-8 h, rising to 80 +/- 4% of MII after 12 h. MAPK activity decreased to a low level 4 h after activation, increased between 6-12 h, but remained below 30 +/- 3.6% of MII values. The incubation with 6-DMAP had no effect on the kinetics of MPF and MAP kinase activity. Fusion of MII oocytes to GV partners induced rapid breakdown of the GV, whereas no breakdown occurred when GV were fused with eggs in the first hours post activation. Interestingly, the high biochemical levels of MPF activity at 8-12 h after activation were not able to induce GVBD in fusion partners.

Adenine↗

PubMed2000

Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis.

The major obstacle in the extensive analysis of the embryonic genome is the small number of cells typically obtained after the embryo biopsy. The object of the present study was to develop a simple approach that would allow the collection of a sufficient number of cells from a single embryo for use in further analyses. A micromanipulator was used to make a hole in the zona pellucida of 28 compacted morulae, 27 early blastocysts and 31 expanded blastocysts. After further culture, the trophoblastic cells, which herniated through this hole, were cut and cultured in vitro for different periods and used for embryo sexing. The results showed that biopsies can be taken successfully from 96.3% of early blastocysts, compared with 67.7% of expanded blastocysts and 71.4% of compacted morulae. The trophoblastic vesicles contained 20.8 +/- 6.7 cells (mean +/- SEM) and, when cultured, formed a confluent monolayer. The sex of cells cultured was assayed by PCR and the 12 lambs born after transfer of biopsied embryos confirmed its 100% accuracy. Moreover, no significant differences were found in the viability rates in vitro among blastocysts vitrified immediately after biopsy (77.8%), blastocysts biopsied and vitrified after 24 h culture (76.9%) and blastocysts vitrified without manipulation (88.5%). In experiments in vivo, the lambing rate of biopsied and vitrified blastocysts was significantly (P < 0.05) lower (40.0%) compared with vitrified control embryos (68.7%). This new approach to the biopsy of preimplantation embryos is a useful good model in the assisted reproductive technologies of domestic, wild and human species.

Animals↗

PubMed1999

Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes.

The aim of this study was to evaluate the effect of gonadotropin treatment on the in vitro maturation, blastocyst production, and developmental potential to term of oocytes collected from Sardinian neonatal and prepubertal ewes at 4 to 6 wk of age. Cumulus-oocyte complexes were recovered at 24 h after withdrawal of a 1/6th size progestagenated pessary from the donors, of which each received 120 IU FSH/LH and 400 IU PMSG in a single dose 36 h before sponge removal. Treated donors produced a greater (P<.01) number of oocytes per animal (86.2 +/-7.9) compared with slaughterhouse (untreated) prepubertal ewes (55.5+/-6.1) of the same age or with treated neonatal ewes (6.1+/-0.7) 10 d old. During oocyte maturation, there were no differences in the percentage of germinal vesicle break-down (78.08 vs. 74.24), metaphase I (89.13 vs. 87.18), and metaphase II (77.91 vs. 76.38) when evaluated after 8, 14, and 24 h of maturation, respectively, between oocytes from treated and slaughterhouse (untreated) prepubertal ewes. The embryo cleavage (71.1 vs. 73.7) and blastocyst rates (22.2 vs. 19.8) were similar in the treated and the untreated prepubertal ewes after transfer of in vitro matured oocytes into ligated oviducts of temporary recipients. The in vitro viability rates of vitrified blastocysts (81.2 vs. 76.9) and the in vivo survival rates (46.1 vs. 50.0) of embryos derived from in vitro matured and in vivo fertilized oocytes showed no difference. The data suggest that gonadotropin treatment increases oocyte production per animal but has no effect on oocyte quality because embryo production and lambing rates of blastocysts derived from in vitro matured oocytes were not markedly different from those derived from untreated prepubertal ewes of the same age.

Animals↗

PubMed1998

Membrane integrity and fertilizing potential of cryopreserved spermatozoa in European mouflon.

There is a pressing need to develop and use assisted reproductive techniques in wildlife species living in small and captive groups. We evaluated the effect of freezing on membrane integrity and fertilizing capacity of European mouflon (Ovis gmelini musimon) spermatozoa collected during the breeding season. After thawing, the percentage of live spermatozoa, stained with fluorescein isothiocynate labeled Pisum Sativum agglutinin and propidium iodide, was 47% of which 19% showed intact acrosomal membrane. After culture in TCM 199 + 10% FCS, the number of live spermatozoa was significantly (P < 0.01) lower than in a medium with oviductal epithelial cells. The absence of oviductal cells decreased significantly the fertilization rates (P < 0.05), 24.0 vs. 63.1 with oviductal epithelial cells and 59.1 in vivo of in vitro matured ovine oocytes. Polyspermic fertilization rate of oocytes was lower (P < 0.05) with oviductal epithelial cells (1.6) than in absence of cells (12.8). However, the percentage of embryos that reached blastocyst stage was significantly higher in vivo than in vitro. These results provide interesting preliminary data for the development of genetic resource banks for European mouflon.

Acrosome↗

PubMed1997

Embryo cloning in sheep: work in progress.

We summarize here the procedures for nuclear transfer using S-phase cytoplasts and describe a new method for avoiding loss of reconstructed embryos from the oviducts during in vivo culture. We obtained 2 clones of 5 genetically identical animals following the transfer of blastomeres from 16-cell embryos into enucleated preactivated cytoplasts. Metaphase II oocytes and embryos were surgically collected from superovulated Sarda breed ewes 54 and 120 h after sponge removal, respectively. Oocytes were exposed for 15 min to 5 mug/ml of Hoechst 33342 and were micromanipulated at room temperature. Efficiency in embryo reconstruction was 100% for enucleation and 98% for fusion. Embryos were embedded in agar as separate clones and transferred into the oviducts of temporary recipients. The fimbriae were closed with glass-nylon made filters. Embryo recovery from the temporary recipients was 97.3%, with a cleavage rate of 81.4%; development to morula-blastocyst stage was 70.6%. A total of 29 Grade 1 blastocysts corresponding to 5 clones were transferred into 13 naturally synchronous ewes, and scanning was performed at 30 and 90 d. Ten ewes were pregnant at the first scanning and nine at the second for a final pregnancy rate of 71.4%; the survival rate at term was 48%. Overall, we obtained 4 clones of identical lambs: two sets of 5 (one male set and one female set) and two sets of twins (both sets male). Pregnancy length in recipients carrying clones was longer than the standard period in Sarda breed (153 vs 150 d, respectively). Weight at birth was higher for male lambs obtained from nuclear transfer than for normal males (4.1 vs 3.6 kg), while the weight for females was normal.

Journal Article↗