Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Artificial insemination of all ejaculated sperm fractions accelerates embryo development and increases the uterine vascularity in the pig.

The semen of boar is characterized by ejaculation in well-differentiated fractions with specific concentration, composition, and volume. The 'sperm-rich fraction' (SRF), the most concentrated seminal fraction, is habitually collected in insemination centers to make artificial insemination (AI) doses. The absence of the other fractions in AI doses could alter the uterine reaction to AI and not trigger essential responses that could maximize fertility. Thus, there is an urge to ascertain the impact of different ejaculate fractions on the uterus after AI to optimize the semen doses. This work analyzed specific parameters related to fertility in pregnant artificially inseminated sows (n = 15) with ac-cumulative fractions of the semen of boars (n = 6): F1, composed of the sperm-rich fraction (SRF); F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF. Non-inseminated sows (n = 5) were included as control (C). The different types of seminal dose did not affect the number of ovulated follicles (CL; corpora lutea, p > 0.05) but did affect the embryo development (p < 0.05). The proportion of embryos in morula stages was significantly higher in AI-F1 sows (84.4%, p < 0.05). Morulas and blastocysts were balanced in AI-F2 or AI-F3 (p > 0.05). Independently of the type of seminal dose (F1, F2, or F3), we observed by immunohistochemistry that AI significantly increased uterine vascularization, although with some anatomical differences. The cranial region of the uterine horns was significantly more vascularized in AI-F1 or AI-F2 sows (26.7 ± 2.3 and 28.6 ± 2.0%, respectively), and AI-F3 showed significantly less vascularization at that point (17.8 ± 1.6%, p < 0.05). To summarize, the synergistic effect of all ejaculate fractions accelerates embryo development, at least during the preimplantation period, and increases the uterine reaction to AI in certain parts of the uterus.

Different seminal ejaculated fractions in artificial insemination condition the protein cargo of oviductal and uterine extracellular vesicles in pig.

The seminal plasma (SP) is the liquid component of semen that facilitates sperm transport through the female genital tract. SP modulates the activity of the ovary, oviductal environment and uterine function during the periovulatory and early pregnancy period. Extracellular vesicles (EVs) secreted in the oviduct (oEVs) and uterus (uEVs) have been shown to influence the expression of endometrial genes that regulate fertilization and early embryo development. In some species, semen is composed of well-separated fractions that vary in concentration of spermatozoa and SP composition and volume. This study aimed to investigate the impact of different accumulative fractions of the porcine ejaculate (F1, composed of the sperm-rich fraction, SRF; F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF) on oEVs and uEVs protein cargo. Six days after the onset of estrus, we determined the oEVs and uEVs size and protein concentration in pregnant sows by artificial insemination (AI-sows) and in non-inseminated sows as control (C-sows). We also identified the main proteins in oEVs and uEVs, in AI-F1, AI-F2, AI-F3, and C-sows. Our results indicated that although the size of EVs is similar between AI- and C-sows, the protein concentration of both oEVs and uEVs was significantly lower in AI-sows (p < 0.05). Proteomic analysis identified 38 unique proteins in oEVs from AI-sows, mainly involved in protein stabilization, glycolytic and carbohydrate processes. The uEVs from AI-sows showed the presence of 43 unique proteins, including already-known fertility-related proteins (EZR, HSPAA901, PDS). We also demonstrated that the protein composition of oEVs and uEVs differed depending on the seminal fraction(s) inseminated (F1, F2, or F3). In conclusion, we found specific protein cargo in oEVs and uEVs according to the type of semen fraction the sow was inseminated with and whose functions these specific EVs proteins are closely associated with reproductive processes.

Lobectomías robóticas y cáncer de pulmón. Experiencia inicial en nuestro centro / Robotic lobectomies and lung cancer. Initial experience in our center

Objetivo: Analizar la seguridad y factibilidad en términos de resultados obtenidos en las primeras lobectomías robóticas realizadas en nuestro centro. Metodología: Estudio prospectivo desde mayo hasta diciembre de 2021 en 13 pacientes (11 hombres y 2 mujeres, edad media 59 años) con carcinoma de pulmón en estadios precoces tributarios de lobectomía robótica.Se utilizó el sistema da Vinci Xi con cuatro puertos y uno asistente. Resultados: Se realizaron 13 lobectomías robóticas. La conversión a cirugía videoasistida fue necesaria en 2 pacientes (15,4%). Se produjeron complicaciones en 3 pacientes (23%). La mediana de tiempo quirúrgico fue180 minutos [IQR 150-210]. La mediana de estancia hospitalaria fue de 4 días [IQR 3 - 6]. La mediana de duración del drenaje pleural fue de 4 días [IQR3 - 6]. La histología predominante fue carcinoma epidermoide en5 pacientes (39%). La media de ganglios linfáticos resecados fue de 15 (IC 95%: 11 - 19) y la de estaciones ganglionares de 5 (IC 95%: 4 - 5). No hubo mortalidad postoperatoria. El estadio postquirúrgico fue IA2 en 4 pacientes (31%), IB en 3 (23%), IIB en 2 (15%), y IIIA en 1 (7%). No se establecen diferencias estadísticamente significativas entre el IMC, el lóbulo resecado y la presencia de complicaciones (p = 0,5; p = 0,2), ni entre el número de ganglios resecados/número de estaciones ganglionares, y el estadio tumoral (p = 0,4; p = 0,9). Conclusiones: La lobectomía robótica con linfadenectomía hiliomediastínica es factible y segura. Es necesaria mayor experiencia y seguimiento a largo plazo para una adecuada evaluación de los resultados postoperatorios, la eficacia oncológica, y la comparación con las vías de abordaje convencionales. (AU)

Objectives: analyze the safety and feasibility in terms of results obtained in the first robotic lobectomies performed in our center. Method: prospective study from May to December 2021 in 13 patients (11 men and 2 women, mean age 59 years) with lung carcinoma in early stages requiring robotic lobectomy. The da Vinci Xi system was used with four ports and one assistant. Results: 13 robotic lobectomies were performed. Conversion to video-assisted surgery was necessary in 2 patients (15.4%). Complications occurred in 3 patients (23%). The median surgical time was 180 minutes [IQR 150-210]. The median hospital stay was 4 days [IQR 3 - 6]. The median duration of pleural drainage was 4 days [IQR3 - 6]. The predominant histology was squamous cell carcinoma in 5 patients (39%). The mean number of lymph nodes resected was 15 (95% CI: 11 - 19) and the number of lymph nodes resected was 5 (95% CI: 4 - 5). There was no postoperative mortality. The postsurgical stage was IA2 in 4 patients (31%), IB in 3 (23%), IIB in 2 (15%), and IIIA in 1 (7%). No statistically significant differences were established between BMI, the resected lobe and the presence of complications (p = 0.5; p = 0.2), nor between the number of resected lymph nodes/number of lymph node stations, and the tumor stage ( p = 0.4; p = 0.9).Conclusions: robotic lobectomy with hiliomediastinal lymphadenectomy is feasible and safe. Greater experience and long-term follow-up are necessary for an adequate evaluation of postoperative results, oncological efficacy, and comparison with conventional approaches. (AU)

Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM.

BACKGROUND: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits. RESULTS: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group. CONCLUSIONS: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

Modelling Particle Transport and Deposition in the Human Healthy and Stented Tracheobronchial Airways.

The main goal of this study is the quantification of the particle transport and deposition within the human airways during light, normal and exercise breathing conditions using the computational fluid dynamics. In particular we presented a comparison between healthy and stented airways. The considered tracheobronchial model is based on the Weibel symmetric model in which we have inserted the Dumon prosthesis at different locations and on the CT-based geometries of a healthy and a stented airway. The results indicate an important redistribution of the particle deposition locations. Local overdoses can be found in the proximal regions of the prostheses, independently of the breathing conditions, of the particle size and of the considered geometry. The presented work is aimed to contribute to the understanding of the particle deposition in the human lung and to improve drug-aerosol therapies. For patients that underwent airways reconstructive surgery, it can give detailed information about the deposition efficiency and it may help targeting specific airways regions.

Boar sperm with defective motility are discriminated in the backflow moments after insemination.

During insemination, a large number of spermatozoa are deposited in the female genital tract, but a very low percentage is able to colonize the site of fertilization. The influx of neutrophils into the uterine lumen and semen reflux (backflow) are known mechanisms that decrease the number of spermatozoa within the uterus. No report has attempted to ascertain whether the backflow is a random or selective process of the spermatozoa. In this work, sows were inseminated using two populations of spermatozoa in the same proportion: (1) unstained spermatozoa with high motility and (2) stained spermatozoa with low, medium, or high motility. Volume, number, and percentage of stained spermatozoa were evaluated in the backflow (collected at 0-15, 16-30, and 31-60 minutes after insemination). This article provides evidence that (1) the motility characteristics of the spermatozoa do not influence the percentage of sows with backflow, the volume and number of spermatozoa in the backflow; (2) the discarding of spermatozoa in the backflow is not specific during the first moments after insemination (0-15 minutes), whereas later (16-60 minutes), spermatozoa with defective motility (low and medium groups) are discarded in a higher proportion than high group in the backflow ([16-30 minutes: low, 85.13 ± 4.32%; medium, 72.99 ± 5.05%; and high, 54.91 ± 2.38%; P < 0.0001; 31-60 minutes: low, 87.16 ± 6.01%; medium, 87.02 ± 4.01%; and high, 59.35 ± 2.86%; P = 0.001]). Spermatozoa with poor motility are discarded in the backflow probably as a selective process, on the part of the female genital tract or as a result of the intrinsic low spermatozoa motility.

Caninos incluidos, tratamiento odontológico. Revisión de la literatura / Unerupted canines, dental treatment. A literature review

El objetivo de este trabajo es realizar una revisión bibliográfica de los últimos 5 años (2006-2011), sobre la situación actual de los caninos incluidos y su tratamiento. Se ha analizado la incidencia, etiología, diagnóstico y factores pronósticos de su alineamiento, así como las terapéuticas de los mismos. Su manejo es de especial importancia, ya que estos dientes tienen un papel fundamental en la apariencia facial, estética dental, desarrollo del arco dental y la oclusión funcional. Se deben diagnosticar mediante una evaluación clínica y radiológica minuciosa además de un examen radiográfico; determinando las posibles complicaciones asociadas y las opciones de tratamiento individualizándolas en cada caso. Se han planteado diferentes formas de manejarlos que van desde los controles periódicos, la prevención de la inclusión con el tratamiento interceptivo, el tratamiento ortodóncico-quirúrgico o la extracción. Antes de iniciar cualquier procedimiento debemos valorar las características individuales de cada paciente, así como la situación y la inclinación del diente para lograr nuestro objetivo (AU)

The aim of this article is to make a bibliographical review, from studies performed in the last 5 years (2006-2011), to analyse the current trends of the unerupted canines in its epidemiology, aetiology, diagnostic methods, prognostic factors in their alignment; and the various treatment options available. It is especially important because of its relevance in the facial appearance, dental aesthetics and functional occlusion. The diagnosis of the unerupted canine should be made through a thorough clinical and radiographic examination, because it helps in determining the best treatment plan and prevention and/or evaluation of possible complications. Different ways to manage such cases have been suggested from periodic controls, preventing the impaction using interceptive treatment, surgical and orthodontic techniques, and lastly, extraction. Thus, before commencing with any treatment, a proper evaluation of the patient characteristics and the characteristics of the canine should be evaluated to achieve the best possible outcome (AU)

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.

Parametros testiculares y caracteristicas morfologicas de los espermatozoides epididimarios obtenidos postmortem en el toro de lidia / Testicular parameters and morphological characteristic of epididymal spermatozoids collected postmortem in the bullfight

En el presente trabajo se han evaluado determinados parámetros testiculares y características morfológicas de los espermatozoides epididimarios obtenidos postmortem en el toro de lidia, teniendo en cuenta la edad, el peso del animal, la circunferencia escrotal, peso y longitud testicular, y sus variables espermáticas. Se analizaron 12 pares de testículos de toros lidiados en una Plaza de Toros, procedentes de doce astados encaste Domecq de dos ganaderías. Una vez llegado el toro al desolladero de la plaza, se midió la circunferencia escrotal, se extrajeron los testículos y se conservaron a una temperatura de 5ºC, previamente identificados. Posteriormente en laboratorio se realizó el pesaje y medición de cada testículo, se diseccionó la cola del epidídimo para conseguir la muestra. Se midió volumen, concentración espermática, motilidad. Por último se realizaron extensiones con la tinción de eosina nigrosina y se valoraron espermatozoides vivos y muertos, células normales, células anormales y acrosomas normales. La muestra obtenida de cada testículo, fue diluida en una proporción 1:2 con un diluyente comercial (Steridyl®), ambos atemperados a 37ºC, antes de realizar las tinciones y las extensiones. Con el fin de estudiar las características espermáticas y los parámetros testiculares de toros de lidia provenientes de dos ganaderías, se realizó un diseño completamente al azar con un arreglo factorial 2x2 (dos ganaderías y dos testículos). Los resultados muestran que hay diferencias significativas (p<0,05), de peso testicular entre la ganadería 1 de 467,33±17,89 y la ganadería 2 de 500,33±16,23. Para la variable circunferencia escrotal existen diferencias significativas (p<0,01) entre la ganadería 1 de 33,5±0,55 y la ganadería 2 de 36,5±1,52; encontrándose la misma diferencia significativa para el peso del testículo derecho e izquierdo, entre la primera ganadería de 295±4,56 y 293,5±4,41, y la segunda de 323,17±14,61 y 321,12±14,93 respectivamente. Para las demás variables evaluadas, concentracíón espermática, la motilidad individual, vivos y muertos, morfoanomalías y acrosomas normales, no se encontraron diferencias estadísticamente significativas (p<0,05). En general, se encontró que en las variables que hubo diferencias estadísticamente significativas, siempre se presentaba entre ganaderías y no tuvo que ver nada el efecto testículo (AU)

In the present work has been evaluated testicular parameters and morphological characteristic different of epididymal spermatozoids collected postmortem in the bullfight as age and weigh animal, scrotal circumference, weigh and long testicular and spermatic variables. Twelve both testicles of bullfight coming from two farms of Domecq has been analyzed. After arrived of bullfight to slaughterhouse the scrotal circumference was measured and the testicles extracted and preserved to 5º C of temperature after yours identification. Afterwards the weigh and measures of each testicle was realized in the laboratory. The epididymal tail was dissectioned for to collect the sample. The spermatic volume and concentration and motility were measured. For last extension of eosin-nigrosin stamp were realized and life and dead spermatozoids, usual and unusual cells and usual acrosomes were determined. Before to stamp, the sample collected of each testicle was diluted 1:2 proportion with commercial diluted (Steridyl®) both moderating to 37oC. In order to study the sperm characteristics and testicular parameters of bullfighting coming from two farms, was a completely random arrangement design 2 x 2 factorial (two farms and two testes). The results show that there are significant differences (p < 0,05), testicular weight between livestock 1 of 467,33±17,89 and livestock 2 of 500,33±16,23. For variable scrotal circumference, there are significant differences (p < 0.01) between livestock 1 of 33, 5±0, 55 and livestock 36,5±1,52; finding the same significant difference for the weight of the right and left testicle between the first breeding of 295±4, 56, 293, 5±4, 41, and the second in 323, 17±14, 61 and 321, 12±14, 93 respectively. For the other variables evaluated, sperm concentration, individual motility, alive and dead, morfoanomalias and normal acrosomas, no statistically significant differences were found (p < 0,05). In general, it was found that in the variables that were statistically significant differences, always presented between farms and did not nothing to the testicle effect (AU)

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.

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.

Evaluation of a cushioned method for centrifugation and processing for freezing boar semen.

The purpose of this investigation was to evaluate the use of an iodixanol cushion during centrifugation on sperm recovery and yield after centrifugation (sperm recovery, sperm motility, viability, membrane lipid disorder, acrosome reaction and ROS generation); and to investigate how this procedure affects sperm function after freezing-thawing (sperm motility, membrane lipid disorder, acrosomal status and homologous in vitro penetration test). The sperm-rich fractions from fertile boars were centrifuged under two centrifugation régimes: 800xg for 10min (standard method) and 1000xg for 20min with an iodixanol (60% w/v) cushion at the bottom of the centrifuge tubes (Cushion method). The highest recovery was achieved using the cushion method (sperm loss for cushion method was 0.50%+/-0.18 versus 2.97%+/-0.43 for standard method, P<0.01) and sperm quality was not significantly affected by the centrifugation régime. The motion parameters (% progressive motility, % motility, VCL, VSL, VAP, ALH, BCF, P<0.05) of frozen-thawed samples showed higher values using the standard method. However, a higher number of viable spermatozoa with lower lipid disorders were found in spermatozoa processed with the cushion method. The in vitro penetration assay showed that the individual boar influenced the parameters studied but there were no differences between the two centrifugation régimes used. Our results support the hypothesis that the proportion of sperm loss in frozen-thawed semen was significantly influenced by the centrifugation régime. Therefore, the iodixanol cushion method is a suitable tool for cryopreservation of boar semen in order to reduce sperm loss without affecting sperm quality.

Effect of sperm preparation method on in vitro fertilization in pigs.

This study was designed to determine the effect of different sperm preparation treatments before IVF on the acrosome reaction, oocyte penetration time, early embryo development and timing of female and male pronucleus formation. Pooled sperm-rich fractions were (i) washed in PBS, (ii) left unwashed, or (iii) layered in a Percoll gradient. In Expt 1, the proportion of acrosome-reacted spermatozoa, determined by staining with fluorescein isothyocyanate-labelled peanut agglutinin lectin and propidium iodide, was highest after treatment with Percoll (P < 0.001). In Expt 2, oocytes matured in vitro were co-cultured with spermatozoa for 2, 4 or 6 h. Attached spermatozoa were then removed and the oocytes were cultured in fresh IVF medium for 16 h. Both sperm treatment and co-culture time were found to affect penetrability and monospermy rates (P < 0.001); spermatozoa treated with Percoll showed fastest oocyte penetration and highest penetrability. In Expt 3, matured oocytes were co-incubated with spermatozoa pretreated by the three above mentioned procedures (i, ii, iii) for 2, 6 and 2 h respectively. Putative zygotes were then washed and transferred to medium NCSU-23 until the blastocyst stage. In this experiment, sperm treatment had a significant effect on the cleavage rate (P < 0.001) and rate of blastocyst formation (P < 0.05); the group treated with Percoll showed the highest rate of blastocyst formation. Finally, in Expt 4, timing of female and male pronucleus formation for each sperm treatment was determined 4, 6 and 8 h after insemination. The time of female and male pronucleus formation was affected by the sperm treatment and was faster for the Percoll group (P < 0.05). The findings of the present study indicate that treatment with Percoll yields the best results in this in vitro pig embryo production system.

Analysis of in vitro fertilizing capacity to evaluate the freezing procedures of boar semen and to predict the subsequent fertility.

A porcine in vitro fertilization (IVF) system and seminal quality parameters of frozen-thawed boar semen were used to assess the effectiveness of two different thawing rates of frozen boar semen, and to address the question of whether differences between fertility of ejaculates could be predicted in a limited field trial. In the first experiment, two thawing procedures were analysed (37 degrees C, 30 s; 50 degrees C, 12 s) and no differences in sperm quality were found. However, when the procedure was 50 degrees C, 12 s the IVF results showed a higher number of sperm per penetrated oocyte and a near 10 points higher rate of pronuclear formation. In the second experiment, the fertility results obtained in the limited field trial show to be efficient enough for application in a commercial use, especially for three of the employed boars (fertility > or = 80%). In this limited study, the conventional seminal parameters are not accurate enough to discriminate good and bad boars in relation to fertility. On the contrary, parameters of in vitro penetrability are more precise to predict subsequent fertilities. As conclusion, the IVF fertilization system seems to be a good tool to evaluate the quality of frozen-thawed boar semen previous to its commercial way, to verify the bank semen storage quality and a good way to assay new sperm freezing procedures, as it is the more precise evaluating method in estimating the potential fertilizing ability.

Effect of in vitrofertilization medium on the acrosome reaction, cortical reaction, zona pellucida hardening and in vitro development in pigs.

Physiological events at the time of fertilization of pig oocytes may differ in vitro depending on the in vitro fertilization (IVF) medium. This hypothesis was tested by in vitro maturation of pig oocytes for 44 h in NCSU-37 medium and thereafter fertilization with frozen-thawed ejaculated spermatozoa. Three different IVF media (TCM-199, Tyrode's albumin lactate pyruvate (TALP) and Tris-buffered medium (TBM)) were used. For the acrosome reaction test, spermatozoa were incubated for 0-150 min in the three IVF media, and the proportion of live acrosome-reacted and acrosome-intact cells was determined by fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA) and propidium iodide (PI) staining. The cortical granule density of oocytes was evaluated by confocal microscopy, 2.5 and 5.0 h after culture in each medium in the presence or absence of spermatozoa. Zona pellucida resistance to pronase digestion was also determined in the same groups. The percentages of penetration, monospermy, male pronucleus formation, cleavage and blastocyst formation, and the number of cells per blastocyst after culture were determined. The results indicate that the acrosome reaction occurred much faster in TBM than in TCM-199 or TALP medium. Continuous cortical granule synthesis was observed in the three media when oocytes were incubated in the absence of spermatozoa. The presence of spermatozoa triggered the cortical reaction in a large proportion of oocytes fertilized in TCM-199 and TALP media. On the basis of the duration of pronase digestion, the zona pellucida of oocytes incubated in TCM-199 was harder (407.7 +/- 35.5 s) than that of oocytes cultured in TALP (235.4 +/- 18.2 s) or TBM (189.1 +/- 16.8 s). No zona pellucida hardening was noted in oocytes after insemination in any of the media. The percentages of penetration and cleavage were higher in oocytes cultured in TCM-199 and TALP than in TBM. The percentage of monospermy was higher in TCM-199 and TBM than in TALP. No effect of the medium was shown on the percentage of blastocyst formation or on the number of cells per blastocyst. In conclusion, the results highlight how differently the fertilization events take place in each IVF medium and how far these IVF media still are from achieving biological properties of gametes close to those observed in the physiological setting.