Cryopreservation of testicular tissue from the dog (Canis familiaris) and wild boar (Sus scrofa) by slow freezing and vitrification: Differences in cryoresistance according to cell type.

Sperm cryopreservation is the most common procedure used to establish germplasm banks for endangered species - but sometimes sperm cells cannot be obtained. In such cases, freezing testicular tissue may be the only option. The testes contains germ cells at different stages of differentiation, including spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa, among which differences in cryoresistance might be expected. The present work compares the viability and DNA integrity of 'rounded' cells, and of elongated spermatids and spermatozoa, from the dog and wild boar, following the cryopreservation of testicular tissue by slow freezing or vitrification. Cell viability was analyzed by PI/SYBR14 staining, and DNA integrity via the TUNEL technique. For wild boar, no significant differences were seen between the two methods with respect to the percentage of viable cells, nor in the percentage of cells with DNA damage. In the dog, the percentage of viable rounded germ cells (65.0 ± 2.4%) was higher (P < 0.05) after vitrification than after slow freezing (45.1 ± 6.7%). No difference was found between the two methods in terms of the viability of elongated cells. For rounded cells, the percentage of intact DNA was greater (P < 0.05) after vitrification (90.5 ± 2.1%) than after slow freezing (42.6 ± 11.0%), while for elongated spermatids and spermatozoa it was higher (P < 0.05) after slow freezing (66.9 ± 6.1%) than after vitrification (50.7 ± 4.5%). Thus, the response to cryopreservation is cell type-, cryopreservation type-, and species-dependent. Vitrification would appear to be the most appropriate method for preserving dog testicular tissue given the associated high cell viability and low degree of DNA fragmentation, while in wild boar, either method might be used.

Synchronous bilateral breast cancer treated with a 3-week hypofractionated radiotherapy schedule: clinical and dosimetric outcomes

Background and purpose Synchronous bilateral breast cancer (SBBC) accounts for 1–3.5% of breast cancer patients. The aim of this study was to evaluate dosimetric issues, clinical outcomes, and acute toxicities for SBBC patients receiving synchronous bilateral hypofractionated radiotherapy (SBHRT) and to compare them with patients treated with synchronous bilateral normofractionated RT schedule (SBNRT). Materials and methods From April 2016 to March 2020, 39 SBBC patients were referred to our institution. Patients were divided according to their prescription dose: Group A: 50 Gy/25fx (fractions), B: 60–64 Gy/25fx, C: 40.05 Gy/15fx; D: 48 Gy/15fx. Toxicity was evaluated using Common Terminology Criteria for Adverse Events (CTCAE)v.5.0. Results 34 patients were finally evaluated. Median follow-up was 24 months for NF schedule and 9 months for HF schedule. In the HF schedule, no acute side-effects > G2 were observed and no dermatitis was reported in 6th month´s assessments. 95% of patients have no evidence of disease and only 1 patient presented local relapse in the first mammography after RT. No distant failures or deaths were observed. Regarding dosimetric issues, the inter-patient average Dmean for the heart was: Group A: 5.0 Gy (4.6–5.5), Group B: 4.4 Gy (4.1–5.4), Group C: 4.8 Gy (4.5–5.1) and Group D: 5.3 Gy (4.4–5.6). For the lungs, the inter-patient average Dmean was: Group A: 10.8 Gy (9.8–12.2), Group B: 11.5 Gy (11.3–12), Group C: 9.8 Gy (9.3–10.5) and Group D: 10.5 Gy (10–11.3). Conclusions This is the first study reporting the safety, feasibility, and tolerability of 40.05 Gy/15fx over 3 weeks for the treatment of SBBC patients. Further study with larger accrual is mandatory (AU)

Synchronous bilateral breast cancer treated with a 3-week hypofractionated radiotherapy schedule: clinical and dosimetric outcomes.

BACKGROUND AND PURPOSE: Synchronous bilateral breast cancer (SBBC) accounts for 1-3.5% of breast cancer patients. The aim of this study was to evaluate dosimetric issues, clinical outcomes, and acute toxicities for SBBC patients receiving synchronous bilateral hypofractionated radiotherapy (SBHRT) and to compare them with patients treated with synchronous bilateral normofractionated RT schedule (SBNRT). MATERIALS AND METHODS: From April 2016 to March 2020, 39 SBBC patients were referred to our institution. Patients were divided according to their prescription dose: Group A: 50 Gy/25fx (fractions), B: 60-64 Gy/25fx, C: 40.05 Gy/15fx; D: 48 Gy/15fx. Toxicity was evaluated using Common Terminology Criteria for Adverse Events (CTCAE)v.5.0. RESULTS: 34 patients were finally evaluated. Median follow-up was 24 months for NF schedule and 9 months for HF schedule. In the HF schedule, no acute side-effects > G2 were observed and no dermatitis was reported in 6th month´s assessments. 95% of patients have no evidence of disease and only 1 patient presented local relapse in the first mammography after RT. No distant failures or deaths were observed. Regarding dosimetric issues, the inter-patient average Dmean for the heart was: Group A: 5.0 Gy (4.6-5.5), Group B: 4.4 Gy (4.1-5.4), Group C: 4.8 Gy (4.5-5.1) and Group D: 5.3 Gy (4.4-5.6). For the lungs, the inter-patient average Dmean was: Group A: 10.8 Gy (9.8-12.2), Group B: 11.5 Gy (11.3-12), Group C: 9.8 Gy (9.3-10.5) and Group D: 10.5 Gy (10-11.3). CONCLUSIONS: This is the first study reporting the safety, feasibility, and tolerability of 40.05 Gy/15fx over 3 weeks for the treatment of SBBC patients. Further study with larger accrual is mandatory.

Arabidopsis ILITHYIA protein is necessary for proper chloroplast biogenesis and root development independent of eIF2α phosphorylation.

One of the main mechanisms blocking translation after stress situations is mediated by phosphorylation of the α-subunit of the eukaryotic initiation factor 2 (eIF2), performed in Arabidopsis by the protein kinase GCN2 which interacts and is activated by ILITHYIA(ILA). ILA is involved in plant immunity and its mutant lines present phenotypes not shared by the gcn2 mutants. The functional link between these two genes remains elusive in plants. In this study, we show that, although both ILA and GCN2 genes are necessary to mediate eIF2α phosphorylation upon treatments with the aromatic amino acid biosynthesis inhibitor glyphosate, their mutants develop distinct root and chloroplast phenotypes. Electron microscopy experiments reveal that ila mutants, but not gcn2, are affected in chloroplast biogenesis, explaining the macroscopic phenotype previously observed for these mutants. ila3 mutants present a complex transcriptional reprogramming affecting defense responses, photosynthesis and protein folding, among others. Double mutant analyses suggest that ILA has a distinct function which is independent of GCN2 and eIF2α phosphorylation. These results suggest that these two genes may have common but also distinct functions in Arabidopsis.

Survival capacity of Mycoplasma agalactiae and Mycoplasma mycoides subsp capri in the diluted semen of goat bucks and their effects on sperm quality.

This study examines the viability of Mycoplasma agalactiae (Ma) and Mycoplasma mycoides subsp capri (Mmc) during 150 minutes of incubation at 37 °C in contaminated diluted semen (DS) doses. The effects of the presence of both microorganisms on sperm viability, motility, and morphology were also examined. In a second experiment, the viability of Ma and its effects on sperm viability were determined in ejaculate samples and skimmed milk semen extender samples. Ma and Mmc were able to survive in DS at concentrations considered infectious, and no significant differences in mean concentrations were detected (7.1 log colony-forming units [CFU]/mL). However, initial concentration of Ma declined (P < 0.05) from 7.5 to 6.9 log CFU/mL and Mmc declined (P < 0.05) from 7.7 to 7.1 log CFU/mL after incubation. Conversely, ejaculate concentrations of Ma increased significantly (from 7.1 to 7.4 log CFU/mL, P < 0.05). These observations suggest that the natural breeding medium is more suitable for Ma than the medium used for artificial insemination (AI). The presence of Mmc slightly reduced sperm viability in the DS (from 21.7% to 16.6%, P < 0.05). The absence of major effects on sperm quality could lead to the unnoticed use of semen contaminated with Ma and Mmc for AI. As both bacteria were able to survive the conditions of ejaculates and semen doses, these findings suggest a risk of venereal transmission of contagious agalactia and support the use of mycoplasma-free semen samples for (AI).

Cross-talk between free and bound spermatozoa to modulate initial sperm:egg ratios at the site of fertilization in the mammalian oviduct.

This essay proposes that highly localized communication between free and bound spermatozoa in the caudal portion of the oviduct acts to regulate the numbers detaching from the epithelium and progressing to the site of fertilization close to the time of ovulation. Low initial sperm:egg ratios are essential for monospermic fertilization. Liberation of surface macromolecules and metabolic prompting from activated spermatozoa, together with altered patterns of sperm movement and dynamic differences in intracellular Ca(2+) ion status between neighboring sperm cells, would influence the progressive release of spermatozoa from the reservoir in the oviduct isthmus. Different intensities of preovulatory epithelial binding, reflecting a range of states in the sperm surface membranes and associated proteins, would provide a further explanation for a chronologically staggered periovulatory detachment of spermatozoa. Intimate sperm-sperm interactions within the confines of the oviduct isthmus offer a sensitive means of fine-tuning the vanguard of competent male gametes reaching the isthmo-ampullary junction.

Experiencia terapéutica con un grupo de niños y sus aportaciones a la consulta psiquiátrica infantil / Group experience with children and its contribution to the individual psychiatric assistance

En la presente comunicación queremos compartir nuestra experiencia grupal y comentar algunos aspectos teórico-prácticos que surgieron durante el desarrollo de la misma, por su valiosa contribución al diagnostico, al tratamiento y al seguimiento de los pacientes. El grupo está compuesto por cuatro pacientes, dos niñas y dos niños, de entre 8 y 10 años, que acuden a la consulta clínica individual habitual para tratamiento y seguimiento por trastornos de conducta. La experiencia tuvo lugar a lo largo de 12 sesiones con una periodicidad quincenal. El trabajo grupal nos permitió observar a cada paciente en su interacción con los otros miembros del grupo facilitándonos un conocimiento más integrado y comprensivo de su patología y de su carácter y proporcionándonos otro lugar de intervención

In the present communication I would like to share some theoretical as well as practical aspects that arose during our group experience because of its valuable contribution to the diagnosis, treatment and the following of the patients. Four kids, two girls and two boys of eight to nine years, complete the group. They also assist to individual sessions of treatment because of several behavioral problems. Our group experience took place during 12 sessions with a periodicity of one every two weeks. Group therapy allows the observation of the patient's interactions with the other members easing a more comprehensive knowledge of his pathology as well as broadening the possibilities of intervention and benefit the monitoring of the process

Spermatozoa and seminal plasma fatty acids as predictors of cryopreservation success.

There is a lack of information about the importance of fatty acid composition of the human sperm membranes and seminal plasma in the cryopreservation procedure. Our aims were to study the possible relationships between the fatty acid composition of human spermatozoa or seminal fluid before freezing, and the sperm quality, measured in terms of viability and motility, before and after freezing-thawing. A further objective of this study was to determine whether the antioxidant capacity (TAC) of the seminal plasma is related to fatty acid (FA) composition and to success of the cryopreservation process. Polyunsaturated fatty acids (PUFA), ω3 PUFAs and docosahexaenoic acid (DHA) in spermatozoa were significantly positively correlated with sperm viability and motility parameters before and after freezing. An inverse relationship was found for monounsaturated (MUFA), ratio ω6/ω3, ratio saturated saturated fatty acids/PUFA (SFA/PUFA) with the seminal parameters. Seminal plasma fatty acid composition was not related to viability. However, motility parameters before and after freezing were related to stearic acid (C18:0) and DHA. TAC in seminal plasma was directly related to PUFA, w3 and DHA. On the other hand, SFA, C22:0, C24:0 and MUFA in seminal plasma were inversely related to the antioxidant capacity. TAC was directly correlated with motion parameters after thawing, We described a significant correlation between the fatty acid composition of the human spermatozoa or seminal plasma and the sperm parameters of the samples after thawing. PUFA, W3 and specially DHA are directly correlated with sperm motility and viability after freezing/thawing, and MUFA was inversely correlated. This means that in the future the fatty acid composition could be used as a predictor of the capacity of cryopreservation. On the other hand, we could design further procedures to modify the lipid composition or/and antioxidant capacity of ejaculate to make it more resistant to the cryopreservation process.

Equine spermatozoa stored in the epididymis for up to 96h at 4°C can be successfully cryopreserved and maintain their fertilization capacity.

After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4°C up to 96h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing-thawing on viability, chromatin condensation, ROS generation, protein tyrosine phosphorylation and heterologous fertilization rate (ICSI and IVF using bovine oocytes) of sperm recovered from the epididymis up to 96h post castration. The average volume (720±159µL) and the concentration (6.5±0.4×10(9) spermatozoa/mL) of sperm recovered from the epididymis were not affected by storage. Sperm viability after refrigeration at 4°C for up to72h was similar (P<0.01). The effect of sperm dilution in the freezing media showed similar values up to 48h, while viability was preserved up to 72h (P<0.01). Cryopreserved spermatozoa show similar viability between different storage times. Chromatin condensation was not affected by storage time; however, incubation for 30min in freezing medium and freezing-thawing process induced an increase in the chromatin decondensation. ROS generation was not affected by storage up to 96h. Epididymal storage did not affect sperm protein tyrosine phosphorylation patterns; although the pattern of phosphorylation changed to strong staining of the equatorial segment when the sperm where capacitated in sperm-TALP. Finally, successful and similar pronuclear formation (analyzed by ICSI) and in vitro penetration (evaluated with bovine zone free oocyte) was observed using cryopreserved sperm obtained from prolong epididymal storage at 4°C. In conclusion, cryopreservation of epididymal stallion sperm stored for up to 72h in the epididymis at 4°C, maintain both viability and ability to fertilize in vitro.

Evaluación de la viabilidad y motilidad espermática en dosis seminales caprinas para el desarrollo de un modelo de contaminación experimental con Mycoplasmaspp / Goat model for experimental mycoplasma contamination of semen: preliminary results

En los últimos años se ha detectado en diversos centros de inseminación artificial la presencia de machos caprinos en los que se han aislado Mycoplasma agalactiae y Mycoplasma mycoides subsp. capri en muestras de semen, sin que presenten ningún síntoma clínico asociado. La capacidad de las bacterias del género Mycoplasma spp. para producir efectos perjudiciales en la calidad espermática es un hecho constatado en diversas especies animales, incluido el hombre. Además, el riesgo de una posible transmisión venérea y afectación de la calidad espermática podría comprometer los programas de mejora genética caprinos fundamentados en la inseminación artificial. El objetivo del presente trabajo es desarrollar un modelo experimental para el estudio del efecto de la contaminación seminal con Mycoplasma spp. en ganado caprino. Para ello, evaluamos la viabilidad y motilidad espermática de dosis seminales, tras dos horas de incubación a 37 ºC en muestras previamente transportadas bajo dos condiciones de temperatura (4-5 °C y 15-16 ºC). Posteriormente se estudió el efecto de la adición del medio PPLO, en el que se preparan los inóculos de Mycoplasma spp., sobre la calidad espermática. El transporte de dosis seminales a 4 ºC ofrece mejores resultados de motilidad total en semen incubado a 37 ºC durante 120 minutos. El medio PPLO no ejerció efecto significativo sobre los porcentajes de espermatozoides vivos o mótiles totales a lo largo de 150 minutos de incubación. Con dicho medio, se pueden obtener valores de viabilidad y motilidad (total y progresiva) espermática superiores al 50% después de 150 y 60 minutos de incubación, respectivamente. En conjunto, este modelo permitiría el desarrollo de contaminaciones experimentales de dosis seminales caprinas con Mycoplasma spp. con el fin de evaluar su efecto sobre la viabilidad y motilidad espermática (AU)

The presence of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in semen samples taken from asymptomatic bucks placed in artificial insemination centres has been confirmed in the last years. The ability of Mycoplasma spp. to cause adverse effects on sperm quality has been also demonstrated in several animal species including humans. In this sense, the risk of venereal transmission and the effects of mycoplasmas on sperm quality could affect goat breed improvement programs based on artificial insemination. The present study was conducted to develop an experimental model useful to study the effect of Mycoplasma spp. in goat semen. We evaluated the viability and motility of seminal doses maintained during two hours at 37 ºC in semen samples previously kept under two temperature conditions (4-5 °C or 15-16 ºC). The effect of PPLO medium in sperm motility, in which Mycoplasma spp. inocula are prepared, was also studied. Motility results registered in semen samples incubated at 37 ºC during 120 minutes are better in seminal doses kept at 4-5 ºC. PPLO medium had no significant effect on live or motile spermatozoa percentages registered after 150 minutes. Sperm viability and motility values (total and progressive) higher than 50% during the 150 and 60 minutes of incubation respectively were obtained using this medium. Overall, the present model is useful to conduct experimental contamination of goat semen doses with Mycoplasma spp. in order to evaluate its effect on spermatic viability and motility (AU)

Two cases of reciprocal chromosomal translocation (4; 7)(p+; q-) (2; 8)(q-; q+) in piglets produced by ICSI.

In this study, the karyotypes of 14 piglets from four different litters produced by intracytoplasmic sperm injection (ICSI) and embryo transfer were analysed. The chromosome analysis was based on a classical cytogenetic examination following the standard protocols of lymphocyte cultures. Two cases of reciprocal translocation [(4; 7)(p+; q-) and (2; 8)(q-; q+)] were detected in two female transgenic piglets. These animals showed neither anatomical nor physiological alterations and had normal growth. To our knowledge, this is the first karyotype study of piglets produced by ICSI.

Effects of centrifugation through three different discontinuous Percoll gradients on boar sperm function.

In this study, different combinations of 2-step, discontinuous gradient centrifugation were used, consisting of three different combinations of isotonic Percoll (45/60, 60/75 and 45/90%) that allowed us to select different sperm subpopulations from fertile and normozoospermic boars. Our objective in this study is to evaluate the effects of centrifugation through three different discontinuous Percoll gradients on sperm function parameters (motility, viability, morphology, acrosome status, chromatin condensation, DNA fragmentation, ROS generation, tyrosine phosphorylation and intracellular calcium concentration) and the sperm penetrating capacity in an IVF system. All the Percoll treatments evaluated increased the percentage of spermatozoa with normal morphology, the proportion of un-damaged DNA, normal chromatin condensation, motion parameters measured by CASA and the percentage of capacitated spermatozoa with tyrosine phosphorylated proteins compared to control group. Finally, the in vitro oocyte penetrating capacity of boar spermatozoa was significantly affected by Percoll centrifugation. All the Percoll treatments increased the penetration rates and mean number of sperm per penetrated oocyte. Despite the efficiency of all three of the sperm treatments tested in selecting spermatozoa with improved sperm parameters and capacity to penetrate oocytes in vitro, the optimum performance of this system was demonstrated after preselecting spermatozoa by centrifugation on a discontinuous 45/90 Percoll gradient. The P45/90 treatment leads to obtain a higher percentage of spermatozoa which develop properly the capacitation process as it was shown measuring tyrosine phosphorylation and intracellular calcium concentration.

Considerations of viscosity in the preliminaries to mammalian fertilisation.

Migration of spermatozoa in the female genital tract will be strongly influenced by the viscosity of the fluids encountered, yet little systematic analysis has been given to such a consideration. This essay reviews the series of milieux confronting a fertilising sperm during its progression to the oviduct ampulla. Two groups are discussed, first those in which ejaculation is into the vagina, second those in which semen enters the uterus during a protracted mating. Viscous glycoprotein secretions that accumulate in the oviduct isthmus of both groups before ovulation are highlighted, as is the environment generated in the ampulla by the post-ovulatory suspension of oocyte(s), cumulus cells and spermatozoa; follicular and peritoneal fluids may also be present. The viscosity of all female tract fluids responds to cyclical variations in temperature, and these exist within the oviduct near the time of ovulation. Gradations in viscosity influence the pattern and strength of sperm flagellar activity and the rate of forward movement. Measurements of sperm motility are currently made in a physiological medium of constant viscosity and temperature, thereby overlooking changes in the female genital tract. A more sophisticated approach might reveal an adequate fertilising potential in a proportion of putatively poor semen samples.

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders.

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47±0.46nmol/10(8) cells. Following semen cryopreservation, GSH decreased to 1.62±0.13nmol/10(8) cells, a 64% reduction (p<0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p<0.01). Addition of 1mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze-thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.

Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa.

This work was designed to study how this ability is affected by different sperm treatments routinely used for in vitro fertilization (IVF) assay. In this study, boar sperm samples from epididymal or ejaculated origin were processed by three different methods: left unwashed (NW group), washed in Dulbecco's phosphate-buffered saline supplemented with 0.1% BSA (BSA group), and washed on a Percoll(®) gradient (PERCOLL group). After preparation of semen samples, changes in motility patterns were studied by CASA, calcium uptake by spectrofluorimetry, and ROS generation, spontaneous acrosome reaction, and lipid disorder by means of flow cytometry. Finally IVF assays were also performed with the different semen samples and penetrability results evaluated at 2 and 4 h post insemination (hpi). Independently of the sperm treatment, epididymal spermatozoa showed higher values of progressive motility, percentage of live cells with low lipid disorder, and penetration ability at 4 hpi than the corresponding ejaculated spermatozoa. Ejaculated spermatozoa showed higher levels of calcium uptake, ROS generation and percentage of spontaneous acrosome reaction than epididymal sperm. Regarding sperm treatments, PERCOLL group showed the highest values for some motility parameters (linearity of the curvilinear trajectory, straightness, and average path velocity/curvilinear velocity), ROS generation and penetration ability at 2 and 4 hpi; however this same group showed the lowest values for sperm curvilinear velocity and lateral head displacement. From all experimental groups, ejaculated-PERCOLL-treated spermatozoa showed the highest fertilization ability after 2 hpi. Results suggest that capacitation pathways can be regulated by suitable treatments making the ejaculated sperm able to reach capacitation and fertilize oocytes in similar levels than epididymal spermatozoa, although most of the studied capacitation-associated changes do not correlate with this ability.

Effects of porcine pre-ovulatory oviductal fluid on boar sperm function.

Sperm storage within the oviductal isthmus prior to ovulation typically involves binding to oviductal epithelial cells, which are thought to modulate sperm functions including internal calcium concentration, membrane fluidity, and motility. Around the time of ovulation the spermatozoa are gradually released so that they eventually encounter the oocytes within the oviductal ampulla. Previous studies have shown that the oviductal epithelial cells selectively sequester high quality spermatozoa, but the role of oviductal fluid as a selective modulator of sperm function has been investigated to a lesser extent. Here we address the hypothesis that oviductal fluid is also likely to modulate sperm function. Using samples of porcine oviductal fluid collected in the follicular phase of the estrus cycle, we show that short exposure (20 min to 50 microg/mL of oviductal fluid proteins) to either of two separate proteins fractions (> or < 100 kDa) promotes boar sperm viability and acrosomal integrity, decreases sperm plasma membrane fluidity (measured using merocyanine S540), and increases zona binding and polyspermy during in vitro fertilization. Exposure to the lower molecular fraction significantly inhibited, but did not abolish, the bicarbonate-induced stimulation of motility. The results show that subpopulations of spermatozoa respond differentially to oviductal fluid, and suggest that exposure to oviductal fluid in vivo could exert a further level of functional sperm selection.

Evaluación de la recombinasa reca sobre la unión espermatozoide-ADN exógeno en la transgénesis mediada por espermatozoides en la especie porcina / Evaluation of boar sperm-RecA: ssDNA complex interaction

La transgénesis mediada por espermatozoides (SMGT) es un método de producción de animales transgénicos.Se basa en la capacidad que presentan los espermatozoides de unir ADN exógeno y transferir el transgén a los ovocitos. Por otro lado, la proteína recombinasa de origen bacteriano RecA protege las cadenas simples de ADN de la degradación, al crear una capa protectora. Este hecho da lugar a una mayor producción de embriones viables e integración del transgén en animales producidos mediante microinyección pronuclear e ICSI. El objetivo de este estudio fue investigar la capacidad de transferencia de los complejos RecA:ssADN en espermatozoides de cerdo, así como la viabilidad de los mismos mediante citometría de fl ujo. En primer lugar, se recolectó el semen y se centrifugó para eliminar el plasma seminal. Se utilizó el plásmido EGFPque fue incubado con los espermatozoides a 16º C (grupo control). Para el grupo RecA, en primer lugar se desnaturalizó el ADN (95ºC, 5 min) y seguidamente, se incubó con la proteína RecA en hielo durante 1h,transcurrido este tiempo estos complejos (RecA:ssADN) formados se incubaron con los espermatozoides a16ºC. La incubación en presencia de la recombinasa RecA supuso un aumento signifi cativo del porcentajede células unidas al ADN con respecto a espermatozoides intactos incubados únicamente con el gen EGFP(AU)

(Control: 19.68±0.73% vs. RecA: 24.69±0.70%; p<0.01). Los resultados mostraron que para ambos gruposla unión se produce principalmente a células no viables (Control: 19.21±0.71% vs. RecA: 24.11±0.69%;p<0.01), y en una muy baja relación a espermatozoides viables (Control: 0.47±0.09% vs. RecA: 0.58±0.09%;p=0.40). Con este estudio hemos demostrado que los complejos RecA:ssADN interaccionan con los espermatozoides porcinos en mayor proporción que para el caso de los espermatozoides únicamente incubadoscon el transgén, y para ambos casos esta asociación se produce principalmente a células no viables. Por lo tanto se podría utilizar la SMGT combinada con técnicas de reproducción asistida como la ICSI para laobtención de embriones y lechones transgénicos(AU)

Sperm mediated gene transfer (SMGT) is an interesting tool for animal transgenesis consisting on theuse of sperm cells as a vector for transmitting exogenous DNA into eggs at the moment of fertilization. Onthe other hand, RecA, a bacterial recombinase, was shown to protect DNA from degradation by creating aprotective coating during its binding to it and resulted in higher embryo survival and transgenic integrationfrequencies in mice produced by ICSI. The objective of this study was to investigate the capacity of transferenceof RecA:ssDNA complex by pig spermatozoa by measuring the sperm DNA-binding ability and viability byfl ow cytometry. Semen was recovered and centrifuged, discarding the seminal plasma. Linealized plasmidDNA was added to sperm and incubated at 16ºC (control group). In RecA group, DNA was denatured (95ºC, 5min) and incubated with RecA on ice for 1h, then mixed with sperm. RecA group sperm signifi cantly increasedDNA-binding capacity compared to control group semen after 120 min (Control: 19.68±0.73% vs. RecA:24.69±0.70%; p<0.01). Exogenous DNA bound mainly to spermatozoa with reduced viability in all the groupsof spermatozoa evaluated (Control: 19.21±0.71% vs. RecA: 24.11±0.69%; p<0.01). In consequence, only a lowpercentage of living spermatozoa was bound to DNA (Control: 0.47±0.09% vs. RecA: 0.58±0.09%; p=0.40).In this study we have demonstrated that the complex RecA:ssDNA bind with the pig sperm in a higher relationthan sperm incubated only with the transgene, and in both cases the binding is associated mainly with nonviablecells. Therefore, the SMGT technique could combines with assisted reproductive techniques such asICSI to obtain embryos and transgenic piglets(AU)

Differing sperm ability to penetrate the oocyte in vivo and in vitro as revealed using colloidal preparations.

The penetration ability of boar (Sus scrofa domestica) spermatozoa exposed to viscous preparations under in vivo and in vitro fertilization conditions has been examined. Experiments involving induced ovulation in prepubertal animals and surgical insemination directly into the oviduct isthmus revealed an advantage of colloidal preparations. Based on within-animal comparisons, the incidence of penetration was 100% using both spermatozoa suspended in a viscous preparation of plant extracts and spermatozoa suspended in a control medium. However, percentages of monospermy were 22.2% in 54 oocytes inseminated with the control suspension compared with 62.5% in 48 oocytes inseminated with the colloidal preparation. An in vitro study involving 355 oocytes from slaughterhouse ovaries inseminated with in vitro-capacitated spermatozoa gave similar percentages of penetrated oocytes for both the control and colloidal suspensions. In this case, however, the percentage of monospermy was 32.7% in the control group compared with 10.6% for spermatozoa suspended in the colloidal preparation. Higher mean numbers of sperm inside the oocytes and higher numbers of sperm bound to the zona pellucida were also observed with the colloidal suspensions. In vitro motility and viability for spermatozoa in the colloidal suspensions were enhanced compared with that of the control group. Lower sperm membrane lipid disorder and reactive oxygen species generation were also observed in the viscous solution. These findings suggest that viscous fluids can enhance the ability of sperm to move, bind, and penetrate the oocyte in vitro, although this influence may be masked in vivo due to the already high viscosity in the oviductal fluid close to the time of ovulation.

Effect of sperm treatment on efficiency of EGFP-expressing porcine embryos produced by ICSI-SMGT.

Intracytoplasmic sperm injection-sperm-mediated gene transfer (ICSI-SMGT) is a useful tool for the production of transgenic mice but is still rather inefficient in farm animals. In the current study, we evaluated the effect of the sperm treatments on the efficiency for producing enhanced green fluorescent protein (EGFP)-expressing pig embryos by ICSI-SMGT. Four different sperm treatments were assayed: (1) fresh (control), (2) frozen-thawing (FT), (3) quick freezing without cryoprotectant agents (QF), and (4) Triton X-100 treatment (TX-100). First, we evaluated the DNA-binding ability and the viability of sperm under the different treatments coincubated with exogenous DNA (EGFP) by flow cytometry. Second, we evaluated the embryo production rate and the efficiency in transgene expression in embryos after using these spermatozoa to fertilize oocytes by ICSI. Sperm treatment significantly increased DNA-binding capacity but reduced sperm viability compared with that of the control group. Treatments damaging the spermatozoa's membranes (QF and TX-100) resulted in a greater capacity of sperm binding exogenous DNA than that after FT treatment (P<0.01). Similar rates of EGFP-expressing embryos were obtained from the control, FT, and TX-100 groups (37.04+/-3.52%, 43.54+/-5.41%, and 29.03+/-8.29%, respectively), but were significantly higher in the QF group (80.43+/-5.91%). These results demonstrate that the integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions between an injected exogenous DNA and the sperm chromatin. However, severe sperm treatments such as QF and TX-100 may damage the sperm nucleus, induce DNA fragmentation, and/or lead to chromosomal breakage with a detrimental effect on further embryonic development.

Class prediction of closely related plant varieties using gene expression profiling.

In recent years, class prediction experiments have been largely developed in cancer research with the aim of classifying unknown samples by examining their expression signature. In natural populations, a significant component of gene expression variability is also heritable. Citrus species are an ideal model to accomplish the study of these questions in plants, due to the existence of varieties derived from somatic mutations that are likely to differ from each other by one or a few point mutations but are phenotypically indistinguishable at early vegetative stages. The small genetic variability existing among these varieties makes molecular markers ineffective in distinguishing genotypes within a particular species. Gene expression profiles have been used to predict mandarin clementine varieties (Citrus clementina Hort. ex Tan.) by means of two independent supervised learning algorithms: Support Vector Machines and Prediction Analysis of Microarrays. The results show that transcriptional variation is variety-dependent in citrus, and supervised clustering methods may correctly assign blind samples to varieties when both training and test samples are under the same experimental conditions.