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.

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.

Paratuberculosis, or Johne's disease, is a disease of domestic and wild ruminants that culminate with a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis. The aim of this work was to evaluate the type of immune response, Th1 or Th2, induced by DNA vaccinations in lambs of Sarda breed. Twenty-five lambs, serum negative for M. paratuberculosis, were selected at birth from equally serum negative mothers. The lambs were inoculated at 5 months of age with three different mycobacterial antigens cloned into a mammalian expression vector as fusion protein with the enhanced green fluorescent protein (pEGFP-N1). The animals were divided in five groups containing each five lambs. Each group was vaccinated as following (A: physiological solution; B: Gudair; C: p-85A-Mav; D: p-85A-BCG; E: p-Hsp65). Immune response was evaluated by measuring the expression of INF-gamma (Th1 type response) and IL-10 (Th2 type response) by real-time PCR. Gene expression was estimated by comparing the results with that of beta-actin. INF-gamma expression level was increased in lambs vaccinated with plasmids codifying mycobacterial antigens, in particular with p-Hsp65, in comparison with the controls suggesting stimulation of a Th1 immune response similar to that supported by natural infection of M. paratuberculosis. Moreover, animals were infected orally with live M. paratuberculosis. Three months after vaccination and again INF-gamma and IL-10 expression was evaluated in order to verify in vivo the protection level of the vaccines. Plasmids p-85A-BCG and p-Hsp65 seem to elicit a stronger protective immune response against M. paratuberculosis by evaluating the expression level of INF-gamma and evaluating the presence of M. paratuberculosis and animal cell organ damage post-mortem.

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.

The inducible gene vgf and its peptide products are relevant to the neuroendocrine regulation of homeostasis and reproduction in rodents. We show here that in the anterior pituitary of female sheep the somatotrope, gonadotrope, and lactotrope/thyrotrope cell populations each expressed vgf mRNA, but displayed a distinct profile of VGF immunoreactive peptides. ProVGF C-terminus and VGF(443-588) immunoreactivities were found in lactotropes and thyrotropes, often in a subcellular location restricted to the Golgi area and suggestive of rapid peptide (or proVGF) release upon biosynthesis, while high molecular weight bands consistent with proVGF were shown in pituitary extracts. Distinct seasonal changes were revealed, proVGF C-terminus immunoreactive cells being largely identified as lactotropes during the summer (83.7 +/- 2.1% (mean +/-s.e.m.) versus 27.0 +/- 1.9% during the winter), as opposed to thyrotropes during the winter (73.0 +/- 1.9% versus 16.3 +/- 2.1% during the summer). Conversely, antisera to peptides adjacent to the 'Arg-Pro-Arg' cleavage site, and to the VGF(553-555) N-terminus of the proVGF-derived peptide V, selectively labeled gonadotropes, indicating processing to small peptides not retaining the proVGF C-terminus in such cells. Finally, a peptide related to the VGF(4-240) region was immunostained in somatotropes, shown in a Western blot as a band of relative molecular mass of approximately 16,000. In conclusion, a complex, endocrine cell-type-specific processing of proVGF was revealed. Further to the known inducibility of vgf mRNA upon a range of stimuli, discreet, selective modulations of VGF-peptide profile/s are suggested, possibly involved in specific neuro/endocrine or modulatory mechanisms.

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.

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. (C) 2004 Wiley-Liss, Inc.

Extending the preservation time of fresh semen is an important goal in artificial insemination programs particularly for ewes in natural oestrus, where insemination periods are longer than for ewes synchronized with hormonal treatments. The aim of this study was to evaluate the effect of the antioxidant TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) on the maintenance in long term storage of ram semen motility and fertility. Semen from Sarda breed rams was diluted in two extenders: sodium citrate buffer with TEMPOL and skimmed milk, used as control. Samples diluted with TEMPOL were cooled at either 15 degrees C or 22 degrees C, while those diluted with skimmed milk were cooled at 15 degrees C. Each sample was divided into four stocks, and stored for different times (5 min, 24, 48 and 72 h). Three aliquots were taken from each stock for every storage period. One was immediately evaluated under microscope; one was used for in vitro fertilization; one was incubated for 2 h in controlled humidified atmosphere (5% CO2, 7% O2 and 88% N2) at 39 degrees C, then evaluated for motility and utilized for in vitro fertilization. Ram semen diluted with media containing TEMPOL demonstrated increased motility, fertility and an improved protective effect when it was stored at 15 degrees C.

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.

Although the potential use of reproductive biotechnologies for safeguarding endangered wildlife species is undoubted, practical efforts have met with limited success to date. In those instances in which modern technologies have been adapted to rescuing rare or endangered species, procedures have been applied piecemeal, and no consistent breeding program based on reproductive biotechnologies has been undertaken. Here we describe for the first time the rescue of an endangered species, the European mouflon (Ovis orientalis musimon), by the application of an integrated package of reproductive biotechnologies. This genetic management extended from the initial collection of gametes, through the in vitro production of embryos and interspecific transfer, to the birth of healthy mouflon offspring. In addition, a genetic resource bank for the European mouflon was established, with cryopreserved sperm, embryos, and somatic cells.

Here we report on the successful reprogramming of nuclei from somatic cells rendered nonviable by heat treatment. Granulosa cells from adult sheep were heated to nonphysiological temperatures (55 degrees C or 75 degrees C) before their nuclei were injected into enucleated metaphase II oocytes. Reprogramming was demonstrated by the capacity of the reconstructed embryos to develop to the blastocyst stage in vitro and into fetuses and viable offspring in suitable foster mothers. To our knowledge, this is the first report of cloned mammalian offspring originating from nonviable cells. In addition, our experiments show that heat-treating donor nuclei destabilizes higher-order features of chromatin (but leaves intact its nucleosomal organization) and results in a high proportion of reconstructed embryos developing to the blastocyst stage and beyond.

Ovine blastocysts were produced by maturation, fertilization and in vitro culture (IVM/IVF/TVC) 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 °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.

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.

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.

Although technically established in mammalian species more than 15 year ago, the efficiency of embryo cloning by nuclear transfer in terms of offspring production has been invariably low. This article reviews a series of interrelated experiments carried out from 1992 to 1997 regarding the co-ordination of nuclear and cytoplasmic events after embryo reconstruction. The experiments were undertaken at the Istituto Zootecnico e Caseario per la Sardegna (IZCS) and the sheep used belonged to the Sardinian dairy breed. The use of enucleated, pre-activated recipient cytoplasts resulted in an increased frequency of development to blastocyst of embryos reconstructed with unsynchronized blastomere nuclei. This increase in development was due to the absence of chromatin damage and unbalanced ploidy in nuclei transferred after the decay of MPF (Maturation Promoting Factor). The combination of synchronous S-phase cytoplast-karyoplast nuclear transfer together with the reduction of embryo losses from the oviduct of temporary recipients allowed for the first time the production of large numbers of clones of genetically identical lambs, thus bringing nuclear transfer closer to practical application. Finally, the last series of experiments described deals with the establishment of an alternative protocol for embryonic nuclear transfer where the combined use of a chemical activating agent, ionomycin, with a protein kinase inhibitor, 6-Dimethylaminopurine, resulted in the highest development rates to blastocyst stage of metaphase II enucleated oocytes reconstructed with embryonic nuclei described so far.

Laparoscopic insemination with frozen-thawed semen is currently used for planned matings in the Sarda breeding programme. In order to find a fast and less intrusive artificial insemination (AI) method that could replace laparoscopic insemination, a field comparison of laparoscopic and transcervical techniques was carried out on 200 mature Sarda ewes. After AI, ewes were assigned to teaser and fertile rams for 2 months. Return rates and cumulative (AI + natural mating) lambing rates were recorded over three subsequent 23-day periods. Lambing rates to AI were significantly different (P < 0.01), and were 62% and 7% respectively for laparoscopic and transcervical AI. Cumulative lambing rates after two further 23-day periods of natural mating were no longer significantly different (P > 0.05) and reached 82% and 74% respectively Ewes with body condition scores at AI higher than 2.75 showed better overall reproductive performance, but not higher pregnancy rate to AI. Plasma cortisol concentrations, sampled twice, before and after AI, were higher (P < 0.01) in the last sample, suggesting a stress response to insemination. Cortisol levels after AI were lower (P < 0.01) for ewes submitted to transcervical rather than laparoscopic insemination (P < 0.01). However, cortisol levels after AI were no greater than those recorded when ewes were restrained in a milking yoke different from that usually employed. Laparoscopic AI was confirmed as the most suitable technique for insemination of frozen semen in the Sarda breeding scheme.

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.

Preliminary experiments carried out on ovine oocytes were designed to establish correlations between activation protocols and subsequent rates of embryonic development. The best activation protocols were thereafter used in studies on ovine parthenogenesis and cloning. The first study established that chemical activators induce pronuclear development at a slightly higher rate than physical activation (ionomycin, 96%; ethanol, 95%; electro activation, 80%). Inhibition of second polar body extrusion and one single pronucleus were observed in the majority of the oocytes (approximately 90%) treated for 3 h with 6-dimethylaminopurine (6-DMAP) following either ionomycin or ethanol activation. While over 80% of these oocytes cleaved after transfer to the oviducts of recipients, progression to the blastocyst stage was higher after ionomycin as compared with ethanol activation (58% vs. 19%). The ionomycin plus 6-DMAP activation protocol was used to produce parthenogenetic blastocysts whose subsequent development was monitored both by ultrasonography and by direct fetal examination. Over 70% of parthenogenotes were viable on Day 21 of pregnancy but dead by Day 25. The effects of 6-DMAP on nuclear remodeling and fetal development of cloned embryos was then investigated. Control cloned embryos underwent nuclear envelope breakdown (NEBD), premature chromatin condensation (PCC), and inhibition of DNA synthesis. By contrast, reconstructed embryos treated with 6-DMAP exhibited intact nuclear membranes, interphase chromatin, and no interference on DNA synthesis. Moreover, cloned embryos developed to blastocyst stage in higher percentage after 6-DMAP treatment (83% vs. 25%). We conclude that ionomycin followed by 6-DMAP incubation yields high percentages of diploid parthenogenetic embryos that develop to Day 25 before dying. Cloned embryos activated by the ionomycin-6-DMAP protocol develop readily to term.