Heterologous in vitro fertility evaluation of cryopreserved Iberian red deer epididymal spermatozoa with zona-intact sheep oocytes and its relationship with the characteristics of thawed spermatozoa.

A heterologous in vitro system, using zona-intact sheep oocytes, was used to evaluate the relationship between sperm factors of Iberian red deer thawed epididymal sperm and the percentage of cleaved oocytes. Epididymal spermatozoa were recovered from six males, diluted with freezing extender and cryopreserved. After thawing sperm motility (SM) and acrosome and membrane integrities were evaluated. Again, these parameters were assessed after incubation in freezing extender at 37 degrees C for 2 h. After cryopreservation the values for SM and acrosome and membrane integrities were high (approximately 80, 80 and 70% respectively). However, these values significantly decreased after incubation (approximately 59, 62 and 47% respectively). Red deer thawed epididymal sperm fertilized zona-intact sheep oocytes, although the percentage of cleaved oocytes was low (approximately 22%). No relationship was found between sperm parameters assessed after thawing and the percentage of cleaved oocytes. Likewise, any sperm parameter evaluated after incubation was assessed in relation to the percentage of cleaved oocytes. However, acrosome and membrane integrities were near to significance (p = 0.06 and p = 0.09 respectively). Then, we conducted a reduced model with these two variables and both were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Thus, acrosome and membrane integrities were related to the percentage of cleaved oocytes negatively and positively respectively. It was concluded that the classical parameters assessed in deer thawed sperm samples can be good predictors of the ability to fertilize zona-intact sheep oocytes.

Effect of coculture with oviduct epithelial cells on viability after transfer of vitrified in vitro produced goat embryos.

This study evaluates the effect of coculture with goat oviduct epithelial cells (GOEC) on the pregnancy rate, embryo survival rate and offspring development after direct transfer of vitrified/thawed caprine in vitro produced (IVP) embryos. Oocytes were recovered from slaughterhouse goat ovaries, matured and inseminated with frozen/thawed capacitated semen, and presumptive zygotes were randomly cultured in synthetic oviduct fluid (SOF) (n=352) or GOEC (n=314). The percentage of cleaved embryos reaching the blastocyst stage was 28% and 20% in SOF and GOEC, respectively (P<0.05). Overall, 26 blastocysts of SOF were transferred freshly in pairs to recipient goats, whereas 58 of SOF and 36 of GOEC were vitrified and transferred directly in pairs to recipient goats after thawing without removal of cryoprotectants or morphological evaluation. The kidding rate was 92% for SOF fresh, 14% for SOF vitrified (P<0.001) and 56% for GOEC vitrified (P<0.05); the difference was also significant between vitrified groups (P<0.01). The embryo survival rate was 62% for SOF fresh, 9% for SOF vitrified (P<0.001) and 33% for GOEC vitrified (P<0.05) with a significant difference between vitrified groups (P<0.01). The results showed that the coculture of IVP goat embryos with GOEC significantly improves the pregnancy and embryo survival rates and leads to the birth of healthy offspring. However, further research using more defined GOEC coculture is required to confirm its capacity to increase the success rate of IVP embryo technology in goat.

Successful use of oviduct epithelial cell coculture for in vitro production of viable red deer (Cervus elaphus) embryos.

Techniques for in vitro production (IVP) of viable embryos have been thoroughly developed in several domestic species in view to improve breeding efficiency. When applied to wild life, these techniques may also help the maintenance of biodiversity through amplification of sparse animals offspring and facilitation of genetic material exchange. During the successive steps of IVP, i.e. oocyte in vitro maturation (IVM), fertilization (IVF) and early embryo development (IVD) to the blastocyst stage, gametes and embryos are faced with unusual environment, including oxidative stress, known to be detrimental to their survival. In the present study, starting from methods developed in domestic species, we have adapted IVP to produce viable red deer embryos. In a first experiment, cumulus cells were removed from in vitro matured oocytes either before or after IVF. The presence of cumulus cells during IVF did not affect final cleavage or development rates. In a second experiment, in vitro matured oocytes were fertilized in the presence of cumulus cells and cultured in SOFaaBSA medium alone or in the presence of ovine oviduct epithelial cell (oOEC) monolayer. Whereas, oviduct cells did not improve the cleavage rate, they significantly increased the rate of embryos reaching the blastocyst stage (from 3 to 25% of total oocytes). Ten blastocysts from oOEC coculture were transferred after freezing and thawing to five recipient hinds and gave rise to three pregnancies. The three pregnant hinds gave birth to three live and normal calves.

Effects of fixation method on image cytometric measurement of DNA content and distribution in cells stained for fluorescence with propidium iodide.

We performed image cytometric measurements of DNA content and distribution on cycling human HCT-8 cells stained for fluorescence with propidium iodide (PI). Seven different fixation protocols were evaluated for stoichiometry of PI staining and for their ability to preserve in vivo chromatin structure. Bimodal integrated optical intensity (IOI) histograms were obtained with all fixation protocols. Increased accessibility of DNA to the dye was evident in increased values of the IOI at the GI peak. The fixatives studied, in order of increasing accessibility to DNA, were Regaud's Boehm-Sprenger, Carnoy's, air-drying, methanol, ethanol, and acetone/methanol. In general, the coefficient of variation of the IOI within the G1 peak was higher for fixatives where DNA is less accessible. Features describing the spatial distribution of stain exhibited dramatic changes for Boehm-Sprenger fixation, which were consistent with the observation that in vivo conformation of chromatin is best preserved with this method.

Reoperation for recurrent temporal lobe epilepsy.

The indications for the risks and outcome of reoperation for medically refractory temporal lobe epilepsy have not been well documented. A retrospective review is presented of 40 patients who underwent reoperation on the temporal lobe for recurrent seizures. The mean patient age at the first operation was 22 +/- 7 years (+/- standard deviation). Electrocorticography during the first operation showed interictal epileptic abnormalities from surface electrodes in 97% of the cases and from depth electrodes in the mesiotemporal structures in 38%. The seizures recurred with the same pattern within 6 months after the first operation in 60% of patients and within 2 years in 90%. Postoperative neuroimaging studies showed residual mesiotemporal structures in all cases. The mean time between the two operations was 5.5 +/- 5 years and the mean patient age at the second operation was 28 +/- 8 years. The second operation involved focal resection of the mesiotemporal structures in 30 cases. The mean postoperative follow-up period was 4.8 +/- 2.7 years (range 2 to 11 years). After the second operation, 63% of the patients were seizure-free or had rare seizures (one or two per year). There were no permanent neurological complications. Patients who did not benefit from reoperation had electroencephalographic abnormalities in multiple brain areas. Reoperation for temporal lobe epilepsy effectively controls seizures in the majority of patients, and the procedure is safe if rigorous technical rules are observed. More complete resection of mesiotemporal structures during the first operation, even in the absence of intraoperative electrographic abnormalities, could prevent the need for reoperation in defined cases.

Current status of embryo technologies in sheep and goat.

This review presents an overview of the technical bases of in vivo and in vitro embryo production in sheep and goat. The current limitations of in vivo production, such as variability of response to the hormonal treatment, fertilization failure in females showing a high ovulatory response, and the importance of premature regressed CL in the goat, are described along with possibilities for improvement. The new prospects offered by in vitro embryo production, by repeated ovum pick-up from live females and by juvenile breeding, are presented along with their limiting steps and research priorities. The recent improvements of embryo production and freezing technologies could be used for constitution of flocks without risks of disease transmission and will allow wider propagation of valuable genes in small ruminants populations in the future.

In vitro fertilization of sheep oocytes matured in vivo.

Incubating washed ram spermatozoa in a modified Brackett's defined medium buffered with Hepes (DM-H) containing 20% of heat-inactivated sheep serum appears to be a reliable method of capacitating sperm for in vitro fertilization. Raising the Ca(++) concentration in the fertilization medium (DM-H-SS) to 10 mM stabilized the fertilization rate of various rams (2). This study was designed to determine if the developmental competence of the oocytes fertilized under such conditions was normal. Thirty-seven ewes, treated with progestagen sponges, were superovulated with porcine follicle stimulating hormone (pFSH: 16 mg). An intramuscular injection of gonadotropin releasing hormone (GnRH: 100 mug); given 24 to 26 h after sponge removal, induced the synchronization of ovulations 24 h later. Ovulated oocytes (n = 229) recovered with flushing of the oviducts were inseminated in vitro and 17 h later either fixed in acetic/alcohol (n = 115) to evaluate fertilization or transferred (n = 114) into 38 synchronized recipients (three oocytes/recipient) to evaluate their developmental competence. Of the fixed oocytes, 82.6% were fertilized and 61.7% were monospermic. Nineteen of the recipient ewes (50%) were pregnant at Day 18, and 16 ewes produced a total of 26 live young (mean: 1.63/ewe). The results showed a high efficiency of in vitro fertilization of ovulated oocytes in sheep following a pFSH-GnRH treatment and the in vivo developmental competence of oocytes fertilized in the presence of elevated Ca(++) concentration.

Effect of growth factors, EGF and IGF-I, and estradiol on in vitro maturation of sheep oocytes.

The objective of these experiments was to determine the effect of exogenous addition of insulin-like growth factor-I (IGF-I, 100 ng/mL), epidermal growth factor (EGF, 10 ng/mL) and estradiol (E2, 100 ng/mL) to the maturation medium of sheep oocytes on their subsequent development in vitro. Addition of IGF-I to the maturation medium did not improve nuclear or cytoplasmic maturation of sheep oocytes at the concentration tested. However, EGF improved significantly the resumption of meiosis (84% oocytes in metaphase II stage after IVM vs. 59% in medium alone). Cleavage rate and blastocyst development rates were improved (P<0.01) after addition of EGF (60% and 29%, respectively), as compared with maturation in TCM 199 alone (39% and 19%, respectively), but remained lower than rates observed after maturation in complete medium containing follicular fluid (FF, 10%) and FSH (81% and 35%, respectively). No additive effect of EGF over FSH was observed during these experiments. Addition of FF to FSH containing maturation medium improved significantly both cleavage (P<0.001) and blastocyst rates (P<0.05). Addition of E2 to the IVM medium is not required when medium already contains FF. However, in defined conditions supplementation of maturation medium with E2 had a positive effect. These results suggest that EGF, FSH and E2 may play an important role in the nuclear and cytoplasmic maturation of sheep oocytes in vitro.

Effect of season and duration of FSH treatment on embryo production in sheep.

Thirty-six mature Manchega ewes were used in two experiments to determine the effect of season and of 2- or 3-d FSHp treatment on the ovulation rate and number of transferable embryos produced. During the breeding season, estrus was synchronized with FGA (30 mg for 13 d). Beginning 48 or 24 h before sponge removal, each ewe received two daily injections of 4-4-3-3-1-1 or 5-5-3-3 mg of FSHp. Concurrently with the two last injections both groups were administered 100 microg of LH. Ewes were tested for estrus and 6 or 7 d later were laparotomized and surgically flushed to recover embryos. The number of corpora lutea (CL), the total number of embryos and of viable embryos were recorded. Six months later (nonbreeding season) the design was repeated, with each ewe receiving the opposite treatment to that received in the fall. Response in ovulation rate and number of viable embryos did not differ between seasons. Mean (SEM) numbers of observed CL and embryos recovered were higher (P<0.001) with the 3-d treatment (8.7+/-5.8 and 7+/-4.8) than with the 2-d treatment (5.8+/-3.2 and 4.4+/-3) when pooled over the two seasons. The mean number of transferable embryos was higher (P<0.01) with the 3-d (4.2+/-3.9) than with the 2-d treatment (2.5+/-2.3).

Can caprine arthritis encephalitis virus (CAEV) be transmitted by in vitro fertilization with experimentally infected sperm?

For each of the five fertilization trials of the experiment, frozen semen was prepared for in vitro capacitation at a concentration of 1 × 10(7) spz/ml and divided into three groups. One group was used as a control, while the two others were inoculated with 100 µl/ml of either culture medium from non-infected cells (placebo group) or cell culture medium containing virus at a concentration of 10(5) TCID(50)/ml (infected group). A total of 789 oocytes were used for IVF. For each of the five trials a group of oocytes were used as a non-infected control and were found to be caprine arthritis-encephalitis virus (CAEV) free. The other oocytes were divided in two equal batches. Oocytes in the first batch were in vitro fertilized with CAEV infected sperm (infected group) and the second batch were fertilized with CAEV non-infected sperm (placebo and control groups). After IVF, the zygotes of each group were washed 12 times. The CAEV genome was not detected (using RT-PCR) in the washing media of either the control or placebo groups from each trial. In contrast, the first three washing media from the infected group were consistently found to be positive for the CAEV genome (5/5), whereas subsequent washing media were CAEV-free (P < 0.05). Zygotes obtained using all semen groups tested negative for both the provirus and genome of CAEV. These results clearly show that the first four washes were sufficient to remove viral particles from CAEV infected fertilization media and that CAEV-free embryos can be produced by IVF using spermatozoa infected in vitro by CAEV.

Maedi-Visna virus was detected in association with virally exposed IVF-produced early ewes embryos.

The objective of this study was to determine whether MVV can be transmitted by ovine embryos produced in vitro and whether the zona pellucida (ZP) provides any protection against MVV infection. Zona pellucida (ZP)-intact and ZP-free embryos, produced in vitro, at the 8-16 cell stage, were cocultured for 72h in an insert over an ovine oviduct epithelial cell (OOEC)-goat synovial membrane (GSM) cell monolayer that had been previously infected with MVV (K1514 strain). The embryos were then washed and transferred to either direct contact or an insert over a fresh GSM cell monolayer for 6 h. The presence of MVV was detected using RT-PCR on the ten washing fluids and by the observation of typical cytopathic effects (CPE) in the GSM cell monolayer, which was cultured for 6 weeks. This experiment was repeated 4 times with the same results: MVV viral RNA was detected using RT-PCR in the first three washing media, while subsequent baths were always negative. Specific cytopathic effects of MVV infection and MVV-proviral DNA were detected in GSM cells that were used as a viral indicator and cocultured in direct contact or as an insert with MVV-exposed ZP-free embryos. However, no signs of MVV infection were detected in cells that were cocultured with exposed ZP-intact or non-exposed embryos. This study clearly demonstrates that (i) in vitro, ZP-free, early ovine embryos, which had been exposed to 10(3) TCID(50)/m MVV in vitro, are capable of transmitting the virus to susceptible GSM target cells, and that (ii) the IETS recommendations for handling in vivo produced bovine embryos (use of ZP-intact embryos without adherent material and performing ten washes) are effective for the elimination of in vitro MVV infection from in vitro produced ovine embryos. The absence of interaction between ZP-intact embryos and MVV suggests that the in vitro produced embryo zona pellucida provides an effective protective barrier.

Quantitative precision of an automated image cytometric system for the measurement of DNA content and distribution in cells labeled with fluorescent nucleic acid stains.

An automated image cytometric system is described for the measurement of DNA content and distribution in cells stained with fluorescent DNA binding or intercalating compounds. The quantitative precision of integrated optical intensity (IOI) measurements using this system was estimated to be 2.0%, based on the coefficient of variation (cv) of the IOI of DNA check beads. Using peripheral blood lymphocytes stained with 4'-6-diamidino-2-phenylindole (DAPI), the cv of IOI was found to be 3.5%. Slide to slide variability of the IOI for peripheral blood lymphocytes (cv over 10 slides of the mean IOI) was found to be 2%. The most important sources of error in these measurements were glare, illumination instability, image calibration instability, and staining non-uniformity. These errors were investigated and minimized.

[Progesterone secretion during male-induced cycle in the Creole goat in anestrus: seasonal effects]. / Sécrétion de progestérone au cours du cycle induit par l'introduction du mâle chez la chèvre créole en anoestrus: effets de la saison.

Plasma progesterone levels during male-induced ovarian cycles were measured at three periods (March, July, November) in Creole goats. For the females experiencing a cycle of normal duration, plasma progesterone reached 2.0 ng/ml six days after male introduction. For the females experiencing a short cycle, a brief (one day) and slight (0.5 to 1.0 ng/ml) secretion of progesterone was observed during this cycle. A seasonal variation in the interval between male introduction and maximum level of progesterone during the short cycle was demonstrated (March: 4.7 days; July: 5.6 d. and November: 3.1 d.), but the maximum level reached did not vary with season (0.6; 0.9 and 0.6 ng/ml).

Nuclear texture measurements in image cytometry.

DNA image cytometry is widely used in cytopathology as a means to obtain objective information concerning the diagnosis and prognosis of human cancer. Using specially designed devices, the high resolution spatial and photometric information is available in the images of a microscopic field. If quantitative DNA specific stains are used the chromatin distribution in the cell nuclei can be measured, which is one of the critical features for cytopathological analysis. In normal cells, changes in the chromatin appearance reflect changes in the activation patterns of genes. In tumors, dramatic changes in the nuclear chromatin appearance are common and have been associated with the progression of the disease. Features describing the chromatin distribution pattern are referred to as texture features. Nuclear texture features are sensitive to the differences between the various descriptive classes of chromatin patterns. In this paper we discuss the main categories of nuclear texture measurements. Texture features can be roughly divided into the following categories: 1) descriptive statistics of chromatin distribution; 2) discrete texture features; 3) range extreme; 4) markovian; 5) run length and 6) fractal texture features. Representative features of each of the above categories are discussed together with mathematical formulas, simple figures for explanation as well as images of typical cells which differ significantly in some texture features. Key references are also provided.

Ovarian capillary blood flow in seasonally anoestrous ewes induced to ovulate by treatment with GnRH.

The microsphere technique was used to obtain estimates of ovarian capillary blood flow near ovulation, in 8 seasonally anoestrous ewes, which were induced to ovulate by GnRH therapy. Plasma progesterone concentrations were monitored in jugular blood sampled between Days 4 and 7 after the onset of the preovulatory LH surge. The ewes were then slaughtered. Three of the ewes were treated with a single injection of 20 mg progesterone before GnRH therapy. In these ewes and 1 other, plasma progesterone values increased after ovulation and reached 1.0 ng/ml on Day 7 following the preovulatory LH surge (normal, functional CL), whilst in the other 4 ewes progesterone concentrations increased initially then declined to 0.5 ng/ml by Day 7 (abnormal CL). In the ewes exhibiting normal luteal function, the mean ovarian capillary blood flow was significantly greater (P less than 0.01) than that for ewes having abnormal luteal function. Irrespective of the type of CL produced, capillary blood flow was significantly greater (P less than 0.05) in ovulatory ovaries than in non-ovulatory ovaries. These findings indicate that the rate of capillary blood flow in ovaries near ovulation may be a critical factor in normal development and maturation of preovulatory follicles and function of subsequently formed CL.

Resumption of oestrous behaviour and cyclic ovarian activity in suckling and non-suckling ewes.

In Préalpes de Sud ewes after an autumn lambing, the mean post-partum interval to first LH surge was 10 +/- 1 days and 17 +/- 1 days for non-suckling and suckling ewes, respectively. Post-partum interval to first luteal phase, estimated from plasma progesterone concentrations, was similar in non-suckling and suckling ewes (27 +/- 1 days and 28 +/- 5 days, respectively). Interval to first oestrus was shorter in non-suckling (22 +/- 2 days) than in suckling ewes (35 +/- 2 days) but these first oestrous periods were followed by short luteal phases in 60% (12/20) of non-suckling ewes and in only 7% (2/29) of suckling ewes. Finally, suckling slightly postponed the resumption of the first oestrus followed by a normal oestrous cycle (37 +/- 1 days versus 31 +/- 2 days) because progesterone, essential for oestrus expression, was secreted mainly during normal luteal phases in 70% (21/30) of suckling ewes and during short cycles in 95% (21/22) of non-suckling ewes. Therefore, the primary consequence of suckling is to regulate the conditions of resumption of cyclic ovarian activity after parturition.

Comparison of DNA measurement performed by flow and image cytometry of embedded breast tissue sections.

Tissue sections are important for morphometric studies, such as the analysis of architectural pattern of tissues as well as nuclear morphology and chromatin texture describing DNA distribution in nuclei. Tissue sections are generally not recommended for use in DNA ploidy measurements due to possible errors based on sectioned and overlapped nuclei. However, image cytometry (IC) on tissue sections has the advantage of preserved architecture and selective sampling of nuclei. This is particularly important for the analysis of specimens with small areas of diseases tissue or with a variety of histologic patterns in the same region, such as premalignant changes or in situ tumors. To evaluate the potential of measurements on breast tissue sections in the estimation of DNA ploidy status, we compared IC and flow cytometry (FC) measurements on formalin-fixed breast cancer tissue from 51 paraffin-embedded blocks. For each specimen the DNA index and ploidy were determined by both methods. DNA indices of IC and FC corresponded roughly in 66% of cases. Agreement in ploidy was achieved in about 80% (40/51) of cases, where some of the discrepancies could be explained by the differences between invasive cancer and ductal carcinoma in situ (DCIS) illustrated by IC. Both methods agreed highly in aneuploid cases. However, about 50% of FC diploid cases were aneuploid by IC. Despite significant shortcomings of measurements obtained on archival tissues, meaningful data can be obtained not only by FC but also by IC performed on tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)

[In vitro oocyte maturation in domestic ruminants]. / Maturation ovocytaire in vitro chez les ruminants domestiques.

In vitro maturation represents the first step towards the in vitro production of embryos in domestic species. This production is of great interest from both zootechnic and basic points of view. Beyond nuclear aspects of maturation (progression of meiosis to metaphase II) cytoplasmic aspects confer to oocytes their potential to be fertilized and to develop normally. The culture medium used for maturation could influence this competence. Furthermore, the origin of oocytes (physiologic and genetic status of the oocyte donor, characteristics of the follicle) could also be determinant. The improvement of in vitro maturation techniques will certainly require a better understanding of these different aspects. Additionally, the maintenance of the oocytes in meiotic block during a pre-maturation in vitro culture will allow them to complete the storage of molecules necessary for fertilization and development.

Differences in quantitative nuclear features between ductal carcinoma in situ (DCIS) with and without accompanying invasive carcinoma in the surrounding breast.

The aim of this study was to demonstrate differences in nuclear morphology between ductal carcinoma in situ (DCIS) without an invasive component and DCIS associated with invasive carcinoma in adjacent breast tissue. DCIS specimens of 60 non-comedo and 21 comedo cases were obtained from two groups of patients with or without invasive carcinoma of the breast. The analysis of DCIS nuclei was performed on formalin fixed deparaffinized thin sections stained with a stoichiometric stain following the Feulgen procedure. Nuclear features, related to nuclear size, shape and DNA distribution, were quantitatively characterized by high resolution image cytometry. Features associated with the presence of invasive carcinoma in the surrounding breast tissue were identified in DCIS nuclei (independent of nuclear grade). Features selected by the stepwise procedure of the discriminant function analysis were typically texture features describing the DNA distribution in the nucleus. A classification function based on the selected nuclear features predicted accurately the presence of invasive carcinoma in all comedo DCIS and in 80% of non-comedo DCIS cases. Our results indicate that quantitative nuclear features of DCIS nuclei are predictive of the accompanying invasion and may be helpful as a new tool in evaluation of DCIS patients.