Changes in the membrane carbohydrates from sperm cryopreserved with dimethylsulfoxide or polyvinylpyrrolidone of red-tailed hawk (Buteo jamaicencis).

BACKGROUND: That cryopreservation can induce alterations in sperm. OBJECTIVE: The goal of this study was to evaluate sperm quality and distribution of N-acetylglucosamine, sialic acid and mannose residues in sperm cryopreserved of red-tailed hawk (Buteo jamaicensis). MATERIALS AND METHODS: We studied twenty samples of ejaculated semen for each cryoprotectant dimethylsulfoxide or polyvinylpyrrolidone. Carbohydrate identification was carried out with lectins Triticum vulgaris agglutinin to N-acetylglucosamine and sialic acid and Concanavalia ensiformis for mannose residues. Sperm viability was not altered but motility decreased significantly with both crioprotectants compared with fresh sperm. RESULTS: Neither the number of WGA positive sperm nor the distribution of N-acetylglucosamine and/or sialic acid residues were affected by the cryopreservation procedure. The sperm proportion with fluorescence associated with the presence of mannose residues was higher in thawed sperm. CONCLUSION: Values obtained with the cryopreservation technique proposed in this study by freezing drops in liquid nitrogen, were within normal parameters established for good quality fresh semen. We can say that it can be used for assisted reproduction of Buteo jamaicensis.

Fertilization capacity of cryopreserved Iberian ibex epididymal sperm in a heterologous in vitro fertilization assay.

In vitro fertilization (IVF) can be used to assess the fertilization capacity of sperm. Heterologous IVF may be useful when assessing that of wild animals as it is often difficult to obtain adequate numbers of naturally corresponding oocytes. The aim of the present study was to assess the fertilization capacity of frozen-thawed ibex epididymal spermatozoa via heterologous IVF involving the oocytes of prepubertal domestic goats. The effect on fertilization and embryo development of adding oestrous sheep serum (ESS) to the fertilization medium was also examined. Cumulus-oocyte complexes (COCs) were matured in TCM-199 for 24-27 h at 38.5°C in a 5% CO2 in air atmosphere. Frozen-thawed epididymal spermatozoa were selected by density gradient centrifugation. After maturation, the oocytes were co-incubated with spermatozoa in synthetic oviductal fluid (SOF) with different concentrations of ESS: SOF-C (0%), SOF-2 (2%) and SOF-20 (20%). At 17 h post-insemination (hpi), zygotes with one female and one male pronucleus (2PN) were categorised as normal; zygotes with 3PN were recorded as polyspermic, and oocytes with 1PN as asynchronous. Cleavage and blastocyst development were assessed at 48 and 168 hpi respectively. The percentage of zygotes with 2PN was higher in the SOF-2 than in the SOF-20 treatment group (27.7% versus 2.9% P < 0.05). The percentage of blastocysts formed with the SOF-C, SOF-2 and SOF-20 treatments were 1.1%, 7.5% and 0% respectively. These results show that the presence of 2% ESS achieves better results than the use of no serum or the standard 20% concentration. Heterologous IVF may be an effective method for predicting the fertilization capacity of ibex spermatozoa, and therefore perhaps that of other wild mountain ungulates.

Sperm characteristics and heterologous in vitro fertilisation capacity of Iberian ibex (Capra pyrenaica) epididymal sperm, frozen in the presence of the enzymatic antioxidant catalase.

The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris-citric acid-glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected: sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P<0.01). The percentage of cleaved embryos at 48hpi was higher (P<0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates.

Diagnostic genetic screening for assisted reproductive technologies patients with macrozoospermia.

Macrozoospermia is characterized by a high proportion of abnormal spermatozoa with enlarged heads. So far, it has been associated with mutations only in the Aurora Kinase C gene (AURKC) in some cases. Although many publications have reported failure to conceive in couples with macrozoospermia, a few others have described successful pregnancies, thus raising questions as to whether ICSI and AURKC genetic screening should be recommended in all patients with macrozoospermia. First, we report on two monozygotic twins presenting macrozoospermia for whom the genetic status was explored (Aurora Kinase C sequencing) and whole semen and gradient-selected spermatozoa were analyzed, using Fluorescent In Situ Hybridization (FISH), Electron Microscopy and flow cytometry. Additionally, FISH analysis was performed on individually selected uniflagellate spermatozoa with normal sized heads. Second, we also provide an updated review of patients with macrozoospermia gathering the percentage of enlarged head spermatozoa, the genetic status and pregnancy outcomes. Both twins carried a homozygous mutation of AURKC. Spermocytograms showed means of 86% and 83.5% of enlarged head forms. FISH analyses showed that normal head size, uniflagellate spermatozoa had an aneuploid or polyploid nucleus despite a high level of selection. SEM analysis also showed special intranuclear inclusions in enlarged head spermatozoa. Our data together with cases reported in the literature allowed us to recommend that the AURKC gene should be sequenced when the sperm contains 30% or more of enlarged head spermatozoa, and when a mutation is found, ART should not be performed. Our analyses provide information that could greatly help practitioners in their decision-making with regard to optimal care of patients with macrozoospermia.

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm.

BACKGROUND: In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up (SU) or swim up + zona pellucida (SU + ZP) binding. RESULTS: Experiment 1, 4-20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN). Treatments showed similar results (54, 47, 42 %, respectively) but statistically differents (P < 0.05) than mechanical activated oocytes in sham, ICSI and sham injection (13, 25, 32 %, respectively) (10-17 replicates; n = 429). Experiment 2: Twelve ejaculates and 28 straws of semen were used (11-19 replicates). Sperm were selected by SU in BSA-TCM 199-H medium. A total of 2,294 fresh sperm and 2,760 from frozen-thawed semen were analyzed after SU or SU + ZP binding. Fresh sperm selected by SU showed acrosome reaction (AR) of 59 %, the sperm selected by SU + ZP binding increased AR to 91 %. In comparison, the AR of frozen-thawed sperm using SU or SU + ZP binding was 77 and 86 %, respectively (P < 0.05). Experiment 3: fertilization in 200 mechanical activativated oocytes (17 replicates) was 4 %, but fertilization increased in ethanol activated oocytes after ICSI (12-28 %) (5-6 replicates). When fresh sperm only selected by SU were injected to 123 oocytes, a fertilization rate (28 %) was achieved; in sperm selected by SU + ZP was 25 % (73 oocytes). In comparison, in frozen-thawed sperm selected by SU, fertilization was 13 % (70 oocytes), whereas sperm from SU + ZP binding displayed 12 % (51 oocytes) (P > 0.05). CONCLUSIONS: Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU + ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU + ZP were significantly different than frozen-thawed sperm, but between sperm treatments no significant differences were obtained.

Detection and Molecular Characterization of Salmonella enterica Serovar Eppendorf Circulating in Chicken Farms in Tunisia.

Salmonella enterica subsp. enterica serovar Eppendorf, with antigenic formula 1,4,12,[27]:d:1,5, is an infrequent serovar. However, 14% (20 of 142) of the isolates recovered during June-July 2012 in chicken farms in Tunisia belonged to S. Eppendorf. These isolates were analysed for resistance and virulence profiles. None of them were susceptible to all antimicrobials tested, while 70%, 60%, 50%, 50%, 20% and 5% were resistant to sulphonamides (sul1, sul2 and sul3), streptomycin (aadA1-like), trimethoprim (dfrA1-like), nalidixic acid (GyrA Asp87 →Asn and not identified), gentamicin (not identified) and ampicillin (blaTEM -1-like). About 30% of the isolates showed decreased susceptibility to ciprofloxacin and carried the qnrB gene; 65% of the isolates were multidrug resistant and contained class 1 integrons with sul1 or sul3 in the 3' conserved segment. The orgA, ssaQ, mgtC, siiD and sopB virulence genes located on SPI1 to SPI5 and the fimbrial bcfC gene were present in all isolates; the sopE1 and sodC1 carried by prophages were variably detected; however, the prophage gipA gene and the spvC gene of serovar-specific virulence plasmids were absent. Altogether, ten resistance and three virulence profiles were identified. Typing of the isolates with XbaI- and BlnI-PFGE supports a close relationship, although they appear to be evolving under selective pressure probably caused by antimicrobial use in chicken husbandry. As far as we know, this is the first study investigating the molecular bases of antimicrobial drug resistance, the virulence gene content and the PFGE profiles of S. Eppendorf. The epidemiological surveillance of this serovar would be necessary to evaluate its possible impact on human health, particularly in Tunisia and other African countries where it was already reported.

Control of a nonholonomic mobile robot using neural networks.

A control structure that makes possible the integration of a kinematic controller and a neural network (NN) computed-torque controller for nonholonomic mobile robots is presented. A combined kinematic/torque control law is developed using backstepping and stability is guaranteed by Lyapunov theory. This control algorithm can be applied to the three basic nonholonomic navigation problems: tracking a reference trajectory, path following, and stabilization about a desired posture. Moreover, the NN controller proposed in this work can deal with unmodeled bounded disturbances and/or unstructured unmodeled dynamics in the vehicle. On-line NN weight tuning algorithms do no require off-line learning yet guarantee small tracking errors and bounded control signals are utilized.

Inhibition of pig oocyte in vitro fertilization by the action of components of the zona pellucida.

The aim of the present study was to determine whether the previous addition of porcine zona pellucida (ZP) components to spermatozoa of the same species has an inhibitory effect on in vitro fertilization (IVF). Boar spermatozoa were exposed to whole porcine solubilized zona pellucida (SZP), ZP glycoproteins (55 kDa and 90 kDa) and peptides (37 kDa, 40 kDa and 68kDa). Doses tested were 40, 70 and 100 mug/ml. In vitro fertilization was clearly inhibited by each component when the oocytes were compared with those fertilized with untreated spermatozoa. All the components had an effect in a dose dependent manner.

Fertility trial in Zebu cattle after a natural or controlled estrus with prostaglandin F2 Alpha, comparing natural mating with artificial insemination.

With the object of comparing reproductive efficiency obtained by natural mating and by artificial insemination (AI), not only following a natural estrus but also after an induced estrus with PGF2Alpha in Zebu cattle in the tropics, 244 adult cows were divided into 4 groups. Group I (N = 69) and Group III (n = 62) were injected with 25 mg of PGF2Alpha when a functional CL was found on rectal examination. Group I was inseminated and group III was served by natural mating, both groups within five days after injection. Groups II (n = 57) and IV (n = 56) were left untreated, group II being AI and group IV ran with a fertile bull for 22 days. Estrus detection was carried out only in the injected groups (I and III) for 15 minutes every three hours between 0600 and 1800. All information was analyzed by linear trigonometric models. The onset of estrus occurred on average 68.7 h after injection in group I and 59.5 h in group III. However only 46.3% and 54.8% of animals were detected in estrus in group I and III respectively, the difference being significant (P < 0.10). Conception rates were 18.6%, 29.8%, 19.3% and 33.9% for groups I, II, III, and IV respectively. A significant difference (P < 0.10) existed between the injected groups and the untreated ones.

Effect of the collection method on semen characteristics of Zebu and European type cattle in the tropics.

The effect of the collection method on the characteristics of fresh semen and the recovery of spermatozoa after thawing was studied in 30 Zebu bulls (Bos indicus) and in 30 Brown Swiss (Bos taurus) bulls. Semen was collected by using an artificial vagina and by electroejaculation; the ejaculates were individually evaluated. Semen was diluted for freezing in skimmed milk and stored in 0.5-ml French straws, at a concentration of 30x10(6) spermatozoa. Data were evaluated by analysis of variance using a factorial model which included collection method, breed effect, and interaction between method and breed, with each bull as a block. Higher volume and pH of the semen was obtained following electroejaculation. Conversely, higher concentration prior to freezing and better progressive motility after thawing was observed in semen collected with an artificial vagina. No differences in motility were obtained in fresh semen between methods. Better post-thaw recovery occurred when the semen was collected by an artificial vagina, independently of the breed type.

Research in animal reproduction: An analysis of the contribution made by Latin America.

In an analysis carried out on the proceedings of the last two international congresses on animal reproduction and artificial insemination (1980 and 1984), a total of 944 papers presented were classified according to sex and species of the experimental animal, reproductive event studied, and country of origin. Latin America was found to be responsible for almost 10% of presentations, with Venezuela and Mexico making up half that total. The bovine species was the most studied both by Latin American countries (51.1%) and in general (42.7%). Approximately 15% of the Latin American contribution was concerned with goat studies versus 3.4% in general. Overall, the female was studied twice as much as the male. Fertility and estrous cycle were the reproductive events most studied both in general (36.3% and 31.6%, respectively) and by Latin American authors (27.2% and 33.3%, respectively). Latin American research concentrated on two main aspects: the effect of environment on reproduction and reproductive efficiency (25.8% and 13.7% of the total, respectively).

Evaluation of the lytic response of prostaglandin F2 alpha in Zebu cattle based on serum progesterone.

To evaluate by means of blood progesterone levels and estrous expression the effect of the injection of 25 mg of PGF2alpha in Zebu cows, 17 Indobrazil cows with a palpable corpus luteum (CL) were injected with PGF2alpha and blood samples taken every 6 h in Experiment I. In Experiment II, 15 cows from the previous experiment at the same stage of diestrus were injected with PGF2alpha and bled every 4 h for 5 d. Progesterone levels had declined by 50% 6 h after injection in all cows and dropped to below 1 ng/ml by 30 h. Estrus was observed in 47% of the animals in Experiment I and 60% in Experiment II. Ovulation was detected accurately in 29% of the animals at 33.6 +/- 11.6 h after the onset of estrus. The correlation between finding a CL by rectal palpation and levels of progesterone higher than 1 ng/ml of blood was 86 and 93% in Experiments I and II, respectively. These studies indicate that the injection of 25 mg of PGF2alpha is sufficient to produce luteolysis in Zebu cattle, although estrous expression is poor.

[Utility of COPA (cuffed oropharyngeal airway) in anesthesia for colonoscopies]. / Utilidad del COPA (cuffed oropharyngeal airway) en anestesia para colonoscopias.

OBJECTIVE: To assess the usefulness of the cuffed oropharyngeal airway (COPA), a new device for airway control, in 45 patients scheduled for colonoscopy. PATIENTS AND METHODS: The patients were anesthetized with propofol and the COPA was applied following the manufacturer's recommendations. Positive pressure ventilation was provided at first, and later the patients were allowed to breathe spontaneously. RESULTS: The mean dose of propofol needed to place the COPA correctly was 2.3 +/- 0.3 mg.kg-1. "Free hands" anesthesia was possible in 43 procedures (96%). Placement had to be attempted several times in five patients (11%) before adequate ventilation was achieved. Two patients (4%) had to be switched to a smaller or larger size COPA. In two others (4%), the technique was abandoned because of inadequate ventilation. No hemodynamic changes were observed after placement, although systolic blood pressure tended to increase slightly during colonoscopy, while heart rate decreased. Spontaneous ventilation was possible in all cases and respiratory frequency and end-tidal CO2 increased significantly during colonoscopy. No cases of laryngospasm or sore throat were observed, although 10 patients (22%) coughed upon emergence from anesthesia. CONCLUSIONS: The COPA is a new alternative to intubation or other methods for controlling the airway during short procedures, making "free hands" anesthesia possible in most cases. Provided contraindications are respected, the number and seriousness of complications seems to be minimal.

Intracytoplasmic sperm injection (ICSI) in small ruminants.

Small ruminants are an important component of the global production systems of meat and wool, and their reproductive biology is well known. However, the incorporation of assisted reproduction techniques (ART) in the production systems of small ruminants is not as well developed as for other domestic species. Normally, production systems that incorporate ARTs are restricted to artificial insemination or in vivo embryo transfer. Intracytoplasmic sperm injection (ICSI) is one of the ARTs techniques reported for small ruminants and consists of the injection of spermatozoa inside an oocyte, bypassing the natural process of sperm-oocyte interaction. In goats and sheep, there are few live births by ICSI reported, with no reports from other species of small ruminants. Currently, there has not been intensive research about ICSI in small ruminants. However, ICSI has potentially important applications in animal production systems, primarily its use with semen of valued animals, with epididymal sperm, in the fertilization of prepubertal or cryopreserved oocytes. Other applications include more advanced techniques, such as transgenic-ICSI or its combination with spermatogonial transplantation. In this article, we review the "state of the art" of this technique in small ruminants including its historical development, research needs for its improvement and future applications.

Individual cryopreservation with dimethyl sulfoxide and polyvinylpyrrolidone of ejaculates and pooled semen of three avian species.

Artificial insemination (AI) has been used for avian reproduction due to the discovery of cryoprotectants extending its usefulness both in production of domestic fowl and conservation of wild species. The goal of this study was to assess the effect on domestic and wild fowl pooled semen and individual ejaculate cryopreservation with dimethyl sulfoxide (DMSO) and polyvinylpyrrolidone (PVP). Twenty ejaculates and twenty samples of pooled semen of roosters, pheasants and hawks were frozen in media containing DMSO or PVP. DMSO and PVP cryopreservation are equally effective both for ejaculates and pooled semen. Even PVP is a good alternative since no significant difference was found when compared to DMSO. The fertilizing capacity of fresh and cryopreserved pooled semen was analyzed through AI of hens and female pheasants. Similar fertility rates using DMSO, PVP or frozen-thawed samples demonstrated that reproduction is possible through the use of cryopreserved semen. In the case of female pheasants, the same values were obtained with both cryopreserved and fresh semen.

Large-sample analysis for a stochastic epidemic model and its parameter estimators.

This paper considers the simplest stochastic model for the spread of an epidemic in a closed, homogeneously mixing population. Approximate methods are presented for calculating the probability distribution of the epidemic size (i.e. number of infected individuals). In fact, a functional central limit theorem and a large deviation principle for the epidemic size when the population increases are shown. These results enable us to both obtain a global approximation for the epidemic size and study asymptotic properties of other random variables depending on the complete history of the epidemic. As an application of our results, we derive two sequences of estimators for the contact rate and analyze their asymptotic behaviour.

Ultrastructural morphology and morphometry of epididymal sperm in the volcano rabbit (Romerolagus diazi).

Sperm characteristics of Romerolagus diazi, an endemic endangered rabbit from Mexico's Higlands, are poorly known. Knowledge of gamete characteristics are urged for any conservation-oriented strategy and morphometry-based taxonomical database. Sperm lagomorph comparisons have been made at light microscopy resolution. Our goal was to analyze the ultrastructure of the R. diazi male gamete. Two wild animals were kept in captivity and the epididymus were obtained. Fixed gametes show a characteristic spatula-like morphology with a dilated forefront. The nucleus has an arrow head morphology lightly thicker at the base. Tail ultrastructure is similar to that of laboratory rabbits with an end piece thicker than that of human sperm. Morphometry data could be used for construction of a male gamete data base for further studies.

Effect of cyproheptadine on chlorpromazine stimulation of prolactin in women.

In five normal women, the expected chlorpromazine-induced rise in serum prolactin was inhibited by pretreatment with a serotonin antagonist, cyproheptadine. This indicated that the serotoninergic system has an excitatory effect on the release of prolactin. When similar studies were done in nine women with hyperprolactinemia and galactorrhea, the administration of chlorpromazine did not consistently produce a rise in serum prolactin, and pretreatment with cyproheptadine did not have a predictable effect. In most cases, this chlorpromazine-induced reduction in the prolactin inhibitory factor could not further increase the secretion of prolactin, so that there was no rise to inhibit.

Expression of IL-2alpha and IL-2beta receptors on the membrane surface of human sperm.

Cytokines are secreted proteins that act as local immunological mediators. Increased seminal cytokine concentrations are associated with fertility problems. The purpose of the present study was to investigate the presence of IL-2alpha, and IL-2beta receptors on fresh and isolated sperm by flow cytometry and transmission electron microscopy. Twenty sperm samples from oligospermic men were incubated with CD25, a mouse monoclonal antibody specific for IL-2alpha-chain receptor, and CD122, a mouse monoclonal antibody specific for IL-2beta-chain receptor. The strong initial fluorescence intensity and, subsequently, a labeling index yielded by CD25 and CD122 decreased in sperm centrifuged on a Percoll gradient (p < .05). The expression of CD25 and CD122 correlated negatively with fresh sperm concentration, but in sperm centrifuged on a Percoll gradient there was no correlation. Labeling with CD25 and CD122 antibody was evident on the head and the middle piece in fresh sperm, while in sperm centrifuged on a Percoll gradient a weak labeling was observed only on the principal piece. The authors have identified and localized cytokine receptors on human sperm for the first time. Cytokine receptors may be involved in the regulation of pathophysiological events in sperm cell functions and male infertility. The exact pathway involved in modulation of these receptors requires further investigation. These results contribute to the understanding of cytokine-sperm relationships.

Acrosome reaction in fertile and subfertile boar sperm.

The main purpose of sperm evaluation is to predict its fertilizing ability. However, basic sperm test results show a low correlation with fertilizing ability. The purpose of this study was to determine whether there is an association between acrosome reaction (AR) and the incidence of subfertility of normal sperm boar. The production records of 22 farms were analyzed to identify boars with low fertility and/or prolificity, classified as subfertile. Twenty-two subfertile boar semen samples were analyzed and compared with 51 samples of fertile boars. Sperm were capacitated during 4 h at 39 degrees C. viability was determined by bisbenzimide (Hoechst-33258) staining. Acrosome reaction was assessed with fluorescein isothiocyanate conjugated Pisum sativum agglutinin. The percentage of spontaneous acrosome reaction (SAR) was not significantly different in fertile (4.5%) and subfertile boars (4.75%) (p > .05). Nevertheless, the percentage of progesterone-induced acrosome reaction (IAR) was significantly lower in subfertile boars (5.75%) as compared with fertile boars (10%) (p < .01). These results suggest that assessment of IAR in vitro may be a useful parameter to identify subfertility in boars.