Ovarian lavage method as an alternative route for hormonal administration and short-term sperm storage in Clarias gariepinus.

Ovarian lavage is a term used to describe the injection of fish with a catheter through the oviduct into the ovary. In this study, the efficacy of this technique was evaluated as a route for hormone administration and sperm preservation in the African catfish Clarias gariepinus. Firstly, the effects of hormone injection routes (namely, intramuscular, intraperitoneal, and ovarian lavage) were evaluated on breeding and haematological parameters. In the second study, the fish's spermatozoa were stored in the ovaries for 1, 2, 3, and 4 days before stripping, sperm activation with freshwater, and fertilization. The breeding performance was then compared with eggs fertilized using spermatozoa refrigerated for the same duration. The study showed that the administration of synthetic hormone (ovaprim®) through the ovaries was comparable to the intramuscular route, while those injected intraperitoneally had the least values (P < 0.05) for breeding performance. The trend of the haematological parameters also suggests the intraperitoneal administration procedure elicited more stress compared to intramuscular or ovarian lavage routes (P < 0.05). In the second experiment, the use of sperm stored in the ovaries or refrigerated for 24hr didn't affect (P > 0.05) the fertilization (92-93%) and hatching (81-83%) of the eggs when compared to the control (91% and 82%). Beyond this 24hr threshold, breeding performances were significantly reduced in the ovarian lavage treatments compared to those fertilized with refrigerated sperm (P < 0.05). The loss of fertilizing capacity observed herein was justified by the reduction in sperm quality over time (P < 0.05). It was concluded that ovarian lavage is a viable route for hormonal administration and 24hr sperm storage in C. gariepinus.

Low levels of exosomal-miRNAs in maternal blood are associated with early pregnancy loss in cloned cattle.

Nuclear reprogramming mediated by somatic cell nuclear transfer (SCNT) has many applications in medicine. However, animal clones show increased rates of abortion and reduced neonatal viability. Herein, we used exosomal-miRNA profiles as a non-invasive biomarker to identify pathological pregnancies. MiRNAs play important roles in cellular proliferation and differentiation during early mammalian development. Thus, the aim of this study was to identify exosomal-miRNAs in maternal blood at 21 days of gestation that could be used for diagnosis and prognosis during early clone pregnancies in cattle. Out of 40 bovine-specific miRNAs, 27 (67.5%) were with low abundance in the C-EPL (Clone - Early pregnancy loss) group compared with the C-LTP (Clone - Late pregnancy) and AI-LTP (Artificial Insemination - Late pregnancy) groups, which had similar miRNAs levels. Bioinformatics analysis of the predicted target genes demonstrated signaling pathways and functional annotation clusters associated with critical biological processes including cell proliferation, differentiation, apoptosis, angiogenesis and embryonic development. In conclusion, our results demonstrate decreased exosomal-miRNAs in maternal blood at 21 days of gestation in cloned cattle pregnancies that failed to reach term. Furthermore, the predicted target genes regulated by these 27 miRNAs are strongly associated with pregnancy establishment and in utero embryonic development.

Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning.

Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine ß-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.

Embryonic development and gene expression in oocytes cultured in vitro in supplemented pre-maturation and maturation media.

This study aimed at assessing the effect of the addition of brain-derived neurotrophic factor (10 ng/ml BDNF) and/or cysteamine (100 µm CYS) during pre-maturation and BDNF, CYS or leptin (10 ng/ml LEP) during maturation culture in vitro on embryo development and oocyte gene expression in cattle. Oocytes were obtained by the aspiration of 2- to 8-mm follicles from slaughtered cows. In Experiment 1, oocytes were pre-matured for 24 h with 10 µm butyrolactone I in the presence or not of BDNF and/or CYS followed by in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 2, oocytes were submitted to IVM with BDNF, CYS or LEP or no supplements followed by IVF and IVC. In Experiment 3, oocytes were pre-matured with BDNF and CYS followed by IVM or only in vitro matured with BDNF. Samples for quantitative PCR (qPCR) were collected after pre-maturation (BGV) and after IVM of pre-matured oocytes (BMII) or immediately after follicle aspiration (immature control = GV) and IVM (matured control = MII). Embryo production was not affected by the inclusion of the different factors either during pre-maturation or maturation culture (∼ 43% blastocysts, p>0.05). Transcripts analysis showed that most genes (NLRP5, ZAR1, GPX1, KEAP1, SPHK2, HSP70 and PSMP1) were downregulated (p<0.05) after IVM irrespective of being previously pre-matured. The relative abundance of BAX, BCL2, IGFBP3 and ARFRP1 transcripts was unaffected by pre-maturation or maturation (p>0.05). In conclusion, supplementation of in vitro pre-maturation (BDNF and/or CYS) or maturation media (BDNF, CYS or LEP) did not improve embryo development. Gene expression was not affected by pre-maturation treatment, but some genes were downregulated after maturation, probably related to selective and differential degradation.

Consequences of nitric oxide synthase inhibition during bovine oocyte maturation on meiosis and embryo development.

The importance of nitric oxide synthase (NOS) in bovine oocyte maturation was investigated. Oocytes were in vitro matured with the NOS inhibitor N(w)-L-nitro-arginine methyl-ester (10(-7), 10(-5) and 10(-3) m L-NAME) and metaphase II (MII) rates and embryo development and quality were assessed. The effect of L-NAME (10(-7) m) during pre-maturation and/or maturation on embryo development and quality was also assessed. L-NAME decreased MII rates (78-82%, p < 0.05) when compared with controls without L-NAME (96%). Cleavage (77-88%, p > 0.05), Day 7 blastocyst rates (34-42%, p > 0.05) and total cell numbers in blastocysts were similar for all groups (146-171 cells, p > 0.05). Day 8 blastocyst TUNEL positive cells (3-4 cells) increased with L-NAME treatment (p < 0.05). For oocytes cultured with L-NAME during pre-maturation and/or maturation, Day 8 blastocyst development (26-34%) and Day 9 hatching rates (15-22%) were similar (p > 0.05) to controls pre-matured and matured without NOS inhibition (33 and 18%, respectively), while total cell numbers (Day 9 hatched blastocysts) increased (264-324 cells, p < 0.05) when compared with the controls (191 cells). TUNEL positive cells increased when NOS was inhibited only during the maturation period (8 cells, p < 0.05) when compared with the other groups (3-4 cells). NO may be involved in meiosis progression to MII and its deficiency during maturation increases apoptosis in embryos produced in vitro. Nitric oxide synthase inhibition during pre-maturation and/or maturation affects embryo quality.

Effect of cyclin-dependent kinase (CDK) inhibition on expression, localization and activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) in bovine oocytes.

This study aimed to assess the effects of cyclin-dependent kinase (CDK) inhibition on factors involved in the control of meiosis in bovine oocytes: maturation promoting factor (MPF) (p34(cdc2) and cyclin B1) and mitogen activated protein kinase (MAPK). Oocytes were maintained at germinal vesicle (GV) stage in vitro with 10 µM of the CDK inhibitor butyrolactone I (BLI) for 24 h (inhibited). After this period, some of the oocytes were transferred to in vitro maturation (IVM) culture for 24 h (inhibited and matured). Control oocytes were assessed immediately after follicle aspiration (immature) or after in vitro maturation for 24 h (matured). Real-time PCR analyses showed that transcripts for p34(cdc2) and MAPK were detected in immature and inhibited oocytes and decreased after maturation, irrespective of CDK inhibition with BLI. Cyclin B1 was detected at similar levels in all oocyte groups. The p34(cdc2) and MAPK proteins were detected by Western blotting at similar levels in all oocyte groups, and cyclin B1 protein was detected only after maturation. Immunofluorescence detection showed that p34(cdc2) was localized in the cytoplasm and GV of immature oocytes, and then throughout the cytoplasm after maturation. Cyclin B1 and MAPK were detected in the cytoplasm in all oocyte groups. Maturation promoting factor and MAPK activities were similar throughout most of maturation for oocytes treated with or without BLI. In conclusion, CDK inhibition did not affect the expression (mRNA and protein levels) and localization of MPF and MAPK, and had nearly no effect on kinase activities during maturation.

Gene silencing during development of in vitro-produced female bovine embryos.

In early development, female embryos (XX) produce twice the transcripts of X-linked genes compared with male embryos (XY). During the course of development, inactivation of the X chromosome equilibrates gene dosage, making the development of female embryos viable. Moreover, the biotechnologies used for producing embryos in vitro seem to work better with male embryos, making it easier for them to reach the blastocyst stage and allow for complete gestation. We investigated the expression of three X-linked genes that are involved in development, XIST, G6PD, and HPRT, and of the transcript interferon-tau, in male and female bovine blastocysts produced by nuclear transfer (NT) and by in vitro fertilization (IVF). Oocytes that had been matured in vitro were enucleated and reconstructed with somatic cells from adult animals at 18 h post-maturation. After fusion (two pulses of 2.25 kv/cm) and chemical activation (5.0 mM ionomycin for 5 min and 2.0 mM 6-DMAP for 3 h), the oocyte-somatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8 degrees C, in a humidified 5% CO(2) atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 10(6) sperm/mL TALP medium supplemented with BSA and PHE and cultivated under the same conditions as the cloned embryos. We used real-time PCR to analyze the gene expression of individual blastocysts compared to expression of the housekeeping gene, GAPDH. The gene XIST was expressed in female embryos and not in male embryos produced by IVF, though it was expressed at low levels in male embryos produced by NT. Unlike previous reports, we found lower levels of the transcript of G6PD in females than in males, suggesting double silencing or other mechanisms of control of this gene. Female embryos produced by IVF expressed the HPRT gene at a higher level than female embryos produced by NT, suggesting that gene silencing proceeds faster in NT-produced female embryos due to "inactivation memory" from the nucleus donor. In conclusion, male and female embryos express different levels of X-chromosome genes and failures of these genes that are essential for development could reduce the viability of females. Nuclear transfer can modify this relation, possibly due to epigenetic memory, leading to frequent failures in nuclear reprogramming.

Unearthing the roles of imprinted genes in the placenta.

Mammalian fetal survival and growth are dependent on a well-established and functional placenta. Although transient, the placenta is the first organ to be formed during pregnancy and is responsible for important functions during development, such as the control of metabolism and fetal nutrition, gas and metabolite exchange, and endocrine control. Epigenetic marks and gene expression patterns in early development play an essential role in embryo and fetal development. Specifically, the epigenetic phenomenon known as genomic imprinting, represented by the non-equivalence of the paternal and maternal genome, may be one of the most important regulatory pathways involved in the development and function of the placenta in eutherian mammals. A lack of pattern or an imprecise pattern of genomic imprinting can lead to either embryonic losses or a disruption in fetal and placental development. Genetically modified animals present a powerful approach for revealing the interplay between gene expression and placental function in vivo and allow a single gene disruption to be analyzed, particularly focusing on its role in placenta function. In this paper, we review the recent transgenic strategies that have been successfully created in order to provide a better understanding of the epigenetic patterns of the placenta, with a special focus on imprinted genes. We summarize a number of phenotypes derived from the genetic manipulation of imprinted genes and other epigenetic modulators in an attempt to demonstrate that gene-targeting studies have contributed considerably to the knowledge of placentation and conceptus development.

Endothelial and inducible nitric oxide synthases in oocytes of cattle.

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (IVM) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM, but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity.

Epaxial and limb muscle activity during swimming and terrestrial stepping in the adult newt, Pleurodeles waltl.

We have investigated the patterns of activation of epaxial musculature during both swimming and overground stepping in an adult newt (Pleurodeles waltl) with the use of electromyographic (EMG) recordings from different sites of the myomeric muscle dorsalis trunci along the body axis. The locomotor patterns of some limb muscles have also been investigated. During swimming, the epaxial myomeres are rhythmically active, with a strict alternation between opposite myomeres located at the same longitudinal site. The pattern of intersegmental coordination consists of three successively initiated waves of EMG activity passing posteriorly along the anterior trunk, the midtrunk, and the posterior trunk, respectively. Swimming is also characterized by a tonic activation of forelimb (dorsalis scapulae and extensor ulnae) and hindlimb (puboischiotibialis and puboischiofemoralis internus) muscles and a rhythmic activation of muscles (latissimus dorsi and caudofemoralis) acting both on limb and body axis. The latter matched the activation pattern of epaxial myomeres at the similar vertebral level. During overground stepping, the midtrunk myomeres express single synchronous bursts whereas the myomeres of the anterior trunk and those of the posterior trunk display a double bursting pattern in the form of two waves of EMG activity propagating in opposite directions. During overground stepping, the limb muscles and muscles acting on both limb and body axis were found to be rhythmically active and usually displayed a double bursting pattern. The main conclusion of this investigation is that the patterns of intersegmental coordination during both swimming and overground stepping in the adult newt are related to the presence of limbs and that they can be considered as hybrid lampreylike patterns. Thus it is hypothesized that, in newt, a chain of coupled segmental oscillatory networks, similar to that which constitutes the central pattern generator (CPG) for swimming in the lamprey, can account for both trunk motor patterns if it is influenced by limb CPGs in a way depending on the locomotor mode. During swimming, the segmental networks located close to the girdles receive extra tonic excitation coming from the limb CPGs, whereas during stepping, the axial CPGs are entrained to some extent by the limb oscillators.

Different forms of impairment of the fore-hindlimb coordination after partial spinal lesions in cats.

Effects of large low thoracic (T10-T11) partial spinal lesions involving either the ventral quadrants of the spinal cord and, to a different extent the dorsolateral funiculi, or different extent of the lateral funiculi and/or the dorsal columns, on the fore-hindlimb coordination were examined in cats walking overground at moderate speeds. In both groups of operated cats, except those in which the lesion was essentially confined to dorsal columns, three different forms of impairment of fore-hindlimb coordination were observed, depending on the extent of lesion: (1) a change of locomotion towards pacing with preservation of the equality rhythms in the fore- and the hindlimbs; (2) episodes of fore- and hindlimb rhythm dissociation and (3) a permanent dissociation of the fore- and hindlimb rhythms. A comparison of the results obtained in these two groups of operated cats points to the more important role played by the lateral funiculi, than by other parts of the spinal white matter, in controlling the fore-hindlimb coordination in cats.

Different patterns of fore-hindlimb coordination during overground locomotion in cats with ventral and lateral spinal lesions.

The effect of large, low thoracic (T10-T11), partial spinal lesions involving the ventral quadrants of the spinal cord and, to a different extent, the dorsolateral funiculi, on fore-hindlimb coordination was examined in cats walking overground at moderate speeds (40-100 cm/s). Three different forms of impairment of fore-hindlimb coordination depending on the extent of the lesions, were observed. Lesions sparing the dorsolateral or the ventral funiculus on one side preserved the equality of the fore- and hindlimb locomotor rhythms but changed the coupling between the movements of both girdles as compared to intact animals. Larger lesions in which, in addition to the ventral quadrants of the spinal cord, also major parts of the dorsolateral funiculi were destroyed elicited episodes of rhythm oscillations in both girdles, which appeared at the background of a small difference in these rhythms. Lesions destroying almost the whole spinal cord induced a permanent difference (about 200 ms) in the step cycle duration of the fore- and the hindlimbs. However, even in these animals some remnant form of fore-hindlimb coordination was found. The results suggest that dorsolateral funiculi play a major role in preserving the equality of rhythms in the fore- and the hindlimbs, while lesions of the ventral quadrants change the coupling between limbs.

Unrestrained walking in cats with medial pontine reticular lesions.

The early postoperative effects of lesions, aimed to destroy the caudal pole of the nucleus reticularis pontis oralis (NRPO) and the rostral pole of the nucleus reticularis pontis caudalis (NRPC), were tested in freely moving cats, walking at moderate speed (0.4-1.0 m/s). In cats in which these structures were partly or completely destroyed, the main effect of lesions was an impairment of fore-hindlimb coordination, as shown by a change in the relationships between the lateral and diagonal time shift durations and the step cycle duration. In the second week after the surgery the values of the slopes of linear regressions relating these variables were markedly changed as compared to the preoperative data. The results suggest that the NRPO and NRPC are involved in maintaining the proper forehindlimb coordination during unrestrained locomotion in cats.

Fictive locomotion in the adult thalamic rat.

In immobilized adult thalamic rats, electrical stimulation of sites within the lateral hypothalamic area (LHA) or the mesencephalic locomotor region (MLR) were found to elicit fictive locomotor patterns in hindlimb muscle nerves. Significant differences were found between several characteristics (average cycle period, locomotor episode duration, intralimb and interlimb coordination patterns) of the LHA-induced and MLR-induced fictive locomotor activities. These findings support the hypothesis that LHA and MLR play different functional roles during locomotion.

Unrestrained walking in intact cats.

In five freely moving cats walking with speeds of 0.4-1.0 m/s several parameters of locomotion were investigated. Special attention was paid to the analysis of support patterns and the duration of support phases. The animals used almost exclusively (in 88 to 99% of steps) the 3-2-3-2-3-2-3-2 support pattern in which phases of support on three limbs alternated with phases of support on two limbs, homolateral and diagonal. The relative duration of support phases showed a tendency to decrease with increased locomotor velocity, except for the supports on diagonal limbs which slightly increased. The mean duration of the majority of support phases was similar and ranged between 12.2 and 14.5% of the step cycle. Phases of support on both hind- and one forelimb were somewhat (about 5%) shorter. It is concluded that the relative stability of support patterns and of the duration of support phases during walking observed in the present experiment may serve as a template for comparing changes in the gait produced by various CNS lesions.

Unrestrained walking in cats with partial spinal lesions.

In four cats with partial spinal lesions, performed at a low thoracic level, involving ventral quadrants and, to a different extent, the dorsolateral funiculi, several parameters of locomotion were analyzed during unrestrained walking at moderate speed (0.3-1.0 m/s). Special attention was paid to the analysis of support patterns and the durations of support phases in step cycles. The operated subjects displayed a much greater variability of support patterns than intact cats as well as changes in the relative duration of some support phases. The most striking difference was an increase in the relative duration of support on two homolateral limbs accompanied by a reduction of support on diagonal limbs. These changes were mainly due to an impairment of fore-hindlimb coordination as shown by an increase in the phase shifts between the movements of diagonal limbs. Other parameters of locomotion were essentially unaltered, except for cats in which the lesion destroyed bilaterally major portions of the dorsolateral funiculi.

Activity of the elbow flexor and extensor muscles during contact placing elicited by tactile stimulation of various aspects of the forelimb in the cat.

The activity of the biceps brachii and lateral head of the triceps brachii were compared during the contact placing reactions elicited by tactile stimuli applied to the lateral, medial or dorsal aspects of the forepaw to verify the hypothesis that common movement strategy was used in all these reactions. Similar latencies and patterns of muscle activation have been found for the medial and dorsal placing while the different timing of the muscle activation was seen in lateral placing reactions. Both muscles often coactivated during lateral or dorsal and less frequently during medial placing reactions. In dorsal placing the coactivations predominated at the beginning of the reactions while in lateral placing they appeared most frequently in later phases of the reaction. The co-contraction of the elbow flexor and extensor muscles leads to locking of the elbow joint. Thus, the coactivation of these muscles in different phases of the lateral, medial and dorsal placing reactions indicates that various movement strategies have been used as the elbow flexion movement is initiated in different phases of these reactions.

Hindlimb swing, stance and step relationships during unrestrained walking in cats with lateral funicular lesion.

In freely moving cats, walking at speeds of 0.4-1.0 m/s, lesions of the lateral funiculi, performed at the low thoracic level, increased the hindlimb step cycle duration and changed the relationships between the stance and swing phase durations and the step cycle duration. The values of the slopes of linear regression lines relating the swing and the step durations were markedly increased, while those for the stance phase were decreased, compared to intact animals. Control dorsal column lesion produced no change in these parameters. The results suggest that pathways in the lateral funiculi play a substantial role in maintaining the proper structure of the step cycle.

Hindlimb muscle activity during unrestrained walking in cats with lesions of the lateral funiculi.

In freely moving intact cats and cats with bilateral lesions of the lateral funiculi the foot contact signals and the activity of selected muscles operating at the ankle and knee joints were analysed during walking at moderate speed (0.4-1.0 m/s). No essential changes in the activity of the muscles gastrocnemius lateralis (GL), semitendinosus (ST) and vastus lateralis (VL) were found in operated animals. The tibialis anterior (TA) muscle activity had a shorter duration than the swing phase in operated animals and showed an impaired coactivation with gastrocnemius lateralis (GL) muscle at the end of the swing phase. Pilot experiments indicated that these deficits may be partly compensated for by peroneal nerve electrostimulation. Analysis of regression lines relating the swing duration to the step duration, determined from EMG records, confirmed our previous results, based on foot contact signals (Górska et al. 1993), showing that in cats with lateral funicular lesions the swing duration varies much more with the step duration than in intact animals.

Locomotion in cats with ventral spinal lesions: support patterns and duration of support phases during unrestrained walking.

The sequences and duration of support phases in the four-legged step cycles performed during unrestrained walking with moderate speed (0.4-1.0 m/s) were analyzed in two groups of cats: intact ones and animals with lesions of the ventral quadrants of the spinal cord at the low thoracic level. Spinal lesions resulted in a much greater variability of support patterns and an increase in the relative durations of the support on two ipsilateral limbs, accompanied by a reduction of support on two diagonal limbs. It is suggested that these changes reflected an impairment of fore-hindlimb coordination, due to an increased phase shift between the onsets of stance phases in the forelimb and in the contralateral hindlimb and may account, at least partly, for the unsteady and ataxic gait described in cats with ventral funicular lesions.