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.

Self-directed learning using computer simulations to study veterinary physiology: Comparing individual and collaborative learning approaches.

BACKGROUND: Advances in technology enable new educational resources geared towards situated learning and leading students to a more active education. Self-directed learning methodologies used along with simulators may represent a good alternative to traditional teaching methods. The aims of this study were to analyse veterinary students' degree of acceptance of self-directed learning using the PhysioEx simulator in physiology, and to evaluate self-directed learning outcomes using different approaches (individual vs. collaborative). METHODS: The study was carried out over three academic years. Students conducted different activities on the PhysioEx simulator, either in an individual or cooperative mode. Once the activities were finished, students voluntarily participated in an opinion survey regarding self-directed learning methodology. Finally, an evaluation of learning outcomes was made through Kahoot!. RESULTS: Students expressed a high degree of satisfaction with this self-directed learning method, with the combination of self-directed learning and gamification being scored the highest. Although students prefer the collaborative method, no differences in learning outcomes were found between the two learning approaches. CONCLUSION: The self-directed learning method in combination with gamification increased the motivation of students, who obtained suitable learning outcomes regardless of whether an individual or collaborative approach was followed.

Evidence of haptoglobin in the porcine female genital tract during oestrous cycle and its effect on in vitro embryo production.

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.

Differential proteome between ejaculate and epididymal sperm represents a key factor for sperm freezability in wild small ruminants.

Epididymal sperm shows higher cryoresistance than ejaculated sperm. Although the sperm proteome seems to affect cell cryoresistance, studies aiming at identifying proteins involved in sperm freezing-tolerance are scarce. The aims of this study were to investigate differences of sperm freezability and proteome between epididymal and ejaculated sperm in three mountain ungulates: Iberian ibex, Mouflon and Chamois. Sperm samples were cryopreserved in straws by slow freezing. Tandem mass tag-labeled peptides from sperm samples were analyzed by high performance liquid chromatography coupled to a mass spectrometer in three technical replicates. The statistical analysis was done using the moderated t-test of the R package limma. Differences of freezability between both types of sperm were associated with differences of the proteome. Overall, epididymal sperm showed higher freezability than ejaculated sperm. Between 1490 and 1883 proteins were quantified in each species and type of sperm sample. Cross species comparisons revealed a total of 76 proteins that were more abundant in epididymal than in ejaculated sperm in the three species of study whereas 3 proteins were more abundant in ejaculated than epididymal sperm in the three species of study (adjusted P < 0.05; |log2| fold-change > 0.5). Many of the proteins that were associated with higher cryoresistance are involved in stress response and redox homeostasis. In conclusion, marked changes of sperm proteome were detected between epididymal and ejaculated sperm. This work contributes to update the sperm proteome of small ruminants and to identify candidate markers of sperm freezability.

Epididymal and ejaculated sperm functionality is regulated differently by periovulatory oviductal fluid in pigs.

BACKGROUND: The current results of in vitro reproduction techniques in pigs, such as in vitro fertilization (IVF) and embryo development, show high performance with both epididymal and ejaculated spermatozoa. However, the results using ejaculated spermatozoa are even better. Ejaculated spermatozoa are exposed to the secretions of the accessory seminal glands: the seminal plasma (SP). It has been reported that exposure of spermatozoa to reproductive fluids, such as SP or periovulatory oviductal fluid (pOF), modulates sperm functionality both in vivo and in vitro. But whether or not this modulating effect of pOF depends on the origin of the spermatozoa being epididymal or ejaculated, is still unknown. OBJECTIVES: To determine and compare the effect of pOF on epididymal and ejaculated sperm functionality. MATERIAL AND METHODS: The effects of incubating spermatozoa from the epididymis and ejaculate with pOF in capacitating conditions were investigated by analyzing sperm motility, phosphorylation of protein kinase A substrates and proteins in tyrosine (pPKAs and pTyr, respectively), the interaction of the spermatozoa with the oocyte in IVF and intracytoplasmic sperm injection (ICSI), and, finally, the spermatozoa chromatin condensation status. RESULTS: The pOF modified events related to capacitation in epididymal spermatozoa by decreasing motility, pPKAs and pTyr. In the interaction with the oocyte after sperm capacitation, pOF regulated the epididymal and ejaculated spermatozoa differently. While pOF decreased the number of spermatozoa bound to the zona pellucida (Spz/ZP) and increased oocyte activation after ICSI with epididymal spermatozoa, with the ejaculated spermatozoa, it decreased the mean number penetrating each oocyte (Spz/O). Additionally, pOF significantly increased the nuclear decondensation of the epididymal spermatozoa after the fertilization of the oocyte. CONCLUSION: The modulation of sperm functionality by pOF is conditioned by the origin of the spermatozoa.

Boar sperm incubation with reduced glutathione (GSH) differentially modulates protein tyrosine phosphorylation patterns and reorganization of calcium in sperm, in vitro fertilization, and embryo development depending on concentrations.

The sperm in the female's reproductive tract undergo changes to fertilize the oocyte (sperm capacitation). These changes are regulated by redox system. However, some assisted reproductive technologies require sperm capacitation under in vitro conditions, though this increases the generation of ROS. Therefore, the aim of this study was to evaluate the effect of GSH as an antioxidant agent during the capacitation of boar sperm [evaluated by calcium compartmentalization, tyrosine phosphorylation (Tyr-P), motility, viability, and acrosomal integrity], in vitro fertilization (evaluated by penetration, monospermy, and efficiency %), and later embryo development (evaluated by cleavage and blastocyst rates, total number of cells per blastocyst and blastocyst diameter). Four experimental groups with different GSH concentrations (0-control, 0.5, 1, and 5 mM) were formed. When 1-GSH was added to the medium, the percentage of capacitated sperm increased after 4 h of incubation; the localization of Tyr-P was modified at 1 h and 4 h of incubation depending on the GSH concentration. Percentages of total and progressive sperm motility also increased at 4 h of incubation, but only in the 5-GSH group compared to control. Viability, acrosomal integrity, and general Tyr-P (Western blot) not differ among the experimental groups. The addition of GSH during gamete interaction increased penetration, monospermy, and efficiency rates in the 1-GSH group compared to the others. However, the effect of GSH was not observed in cleavage and blastocyst rates compared to the control. In conclusion, adding GSH modulates sperm capacitation (by means of calcium compartmentalization and tyrosine phosphorilation pattern) depending on its concentration, and improves IVF output at 1-GSH during gamete interaction.

Epididymal and ejaculated sperm differ on their response to the cryopreservation and capacitation processes in mouflon (Ovis musimon).

Spermatozoa must undergo the process of capacitation to fertilize the egg which involves a cell destabilizing process. Capacitation-like changes such as protein tyrosine phosphorylation (PTP) are associated with cryopreservation. The aim of this study was to compare the cryoresistance and capacitation response of epididymal and ejaculated sperm of European mouflon (Ovis musimon). Post-thaw sperm parameters were analysed from epididymal and ejaculated samples cryopreserved by slow-freezing or ultrarapid-freezing for comparison. Sperm capacitation status was assessed by the semiquantification of PTP levels, cell localization of PTP and kinematic clustering. Epididymal sperm had higher cryoresistance than ejaculated sperm in both freezing techniques, and slow-freezing rendered better results than ultrarapid-freezing in both sperm samples. Ejaculated sperm had higher PTP levels than epididymal sperm and, additionally, ejaculated sperm showed higher phosphorylation in capacitating (CA) than in non-capacitating (NCA) conditions while there was no effect of medium in epididymal sperm. There was a higher tail PTP in CA than in NCA conditions in both types of sperm. Kinematic analysis revealed that the cluster associated with hyperactivated movement increased in ejaculated sperm incubated in CA whereas no effect of medium was observed in epididymal sperm clusters. In conclusion, epididymal sperm showed better freezability and lower capacitation status compared to ejaculated sperm.

Transient Sperm Starvation Improves the Outcome of Assisted Reproductive Technologies.

To become fertile, mammalian sperm must undergo a series of biochemical and physiological changes known as capacitation. These changes involve crosstalk between metabolic and signaling pathways and can be recapitulated in vitro. In this work, sperm were incubated in the absence of exogenous nutrients (starved) until they were no longer able to move. Once immotile, energy substrates were added back to the media and sperm motility was rescued. Following rescue, a significantly higher percentage of starved sperm attained hyperactivated motility and displayed increased ability to fertilize in vitro when compared with sperm persistently incubated in standard capacitation media. Remarkably, the effects of this treatment continue beyond fertilization as starved and rescued sperm promoted higher rates of embryo development, and once transferred to pseudo-pregnant females, blastocysts derived from treated sperm produced significantly more pups. In addition, the starvation and rescue protocol increased fertilization and embryo development rates in sperm from a severely sub-fertile mouse model, and when combined with temporal increase in Ca2+ ion levels, this methodology significantly improved fertilization and embryo development rates in sperm of sterile CatSper1 KO mice model. Intracytoplasmic sperm injection (ICSI) does not work in the agriculturally relevant bovine system. Here, we show that transient nutrient starvation of bovine sperm significantly enhanced ICSI success in this species. These data reveal that the conditions under which sperm are treated impact post-fertilization development and suggest that this "starvation and rescue method" can be used to improve assisted reproductive technologies (ARTs) in other mammalian species, including humans.

Seasonal variation in sperm freezability associated with changes in testicular germinal epithelium in domestic (Ovis aries) and wild (Ovis musimon) sheep.

The aim of this study was to examine ovine sperm cryoresistance during the rutting season (RS) and its association with sperm head area and seminiferous epithelium proliferation. Small ruminants show fluctuating testosterone levels throughout the year, which could interfere with spermatogenesis and sperm cryopreservation. Ejaculates, testicular biopsies and blood were collected during the middle and at the end of the RS (Middle-RS vs End-RS) during periods of high and low testosterone levels in Merino and Mouflon rams. Fresh and frozen-thawed sperm quality, sperm morphometry, seminiferous tubule morphometry and testicular proliferation markers (proliferating cell nuclear antigen, proliferation marker protein Ki-67 and transcription factor GATA-4) were evaluated. Post-thaw sperm viability was higher in the End-RS group in both Merino (69.9±8.2 vs 41.6±7.3%; P=0.020) and Mouflon rams (40.9±3.3 vs 24.2±5.0%; P=0.008). Mouflons had larger sperm head area at the End-RS (38.3±0.2 vs 34.3±0.1µm2; P=0.029), whereas there was no difference between Merino groups (35.7±0.5 vs 34.8±1.0µm2). Seminiferous tubule morphometry and proliferation markers showed higher levels of germinal epithelium proliferation in the Middle-RS of both species. In conclusion, sperm freezability is affected during the RS in domestic and wild rams, which could be correlated with changes that occur during spermatogenesis, since there is an effect of season on cell proliferation in the testis.

Manipulation of bicarbonate concentration in sperm capacitation media improvesin vitro fertilisation output in porcine species.

BACKGROUND: The in vivo concentration of bicarbonate (HCO3 -), one of the essential sperm capacitating effectors, varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of 25 mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3 - concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased. RESULTS: Once exposed to the capacitation medium, the intracellular pH (pHi) of spermatozoa increased immediately even at low concentrations of HCO3 -, but only extracellular concentrations of and above 15 mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15 mmol/L of HCO3 - stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25 mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3 - concentration to 15 mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%). CONCLUSIONS: Optimising HCO3 - concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15 mmol/L of HCO3 - in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcine.

A new device for deep cervical artificial insemination in gilts reduces the number of sperm per dose without impairing final reproductive performance.

BACKGROUND: The aim of this study was to evaluate the reproductive performance of a new artificial insemination (AI) device specifically designed for gilts (Deep cervical AI, Dp-CAI) by means of which the sperm is deposited deeply in the cervix (8 cm more cranial than in traditional cervical insemination-CAI). New AI techniques have arisen in recent decades in the porcine industry, such as post-cervical artificial insemination (PCAI), which involves depositing the sperm in the body of the uterus [through a catheter (outer tube)-cannula (inner tube)] rather than by CAI. Although the PCAI method has been successfully applied in farm conditions to reduce sperm doses without impairing the reproductive performance, this technique has limitations in gilts mainly because of the difficulty involved in introducing the inner cannula through the cranial part of the cervix. For this reason, the Dp-CAI method described herein may be considered as an alternative to CAI and PCAI methods in gilts. RESULTS: Gilts were divided in two experimental groups: 1) Dp-CAI: gilts (n = 1166) inseminated using 1.5 × 109 sperm/45 mL; 2) CAI (as a control group): gilts (n = 130) inseminated using 2.5 × 109 sperm/85 mL. The Dp-CAI method was successfully applied in 88.90% of the gilts, with no differences detected between gilts with 1 or 2 previous oestrus cycles, although the catheter could be introduced more deeply in 2 oestrus gilts (P < 0.05). As the length of the insemination device that could not be introduced increased (at the moment of insemination), so the success rate of the Dp-CAI device fell, as did the total number of piglets born. When the reproductive output in CAI and Dp-CAI was compared, none of the parameters analysed [pregnancy and farrowing rates (%), and number of piglets born (total and live)] showed significant differences. CONCLUSIONS: The use of the Dp-CAI technique provides a new AI method as an alternative to CAI and PCAI for pigs. The device, especially designed for gilts, was used with a high degree of success reducing conventional sperm doses without impairing reproductive parameters.

Tissue plasminogen activator (tPA) of paternal origin is necessary for the success of in vitro but not of in vivo fertilisation in the mouse.

Besides its fibrinolytic function, the plasminogen-plasmin (PLG-PLA) system is also involved in fertilisation, where plasminogen activators bind to plasminogen to produce plasmin, which modulates sperm binding to the zona pellucida. However, controversy exists, depending on the species, concerning the role of the different components of the system. This study focused its attention on the role of the PLG-PLA system on fertilisation in the mouse with special attention to tissue plasminogen activator (tPA). The presence of exogenous plasminogen reduced invitro fertilisation (IVF) rates and this decline was attenuated by the presence of plasmin inhibitors in combination with plasminogen. The incubation of spermatozoa with either oocytes or cumulus cells together with plasminogen did not change the acrosome reaction but reduced the number of spermatozoa attached. When spermatozoa from tPA-/- mice were used, the IVF rate decreased drastically, although the addition of exogenous tPA during gamete co-incubation under invitro conditions increased fertilisation success. Moreover, fertility could not be restored after invivo insemination of tPA-/- spermatozoa in the female ampulla, although tPA-/- males were able to fertilise invivo. This study suggests a regulatory role of the PLG-PLA system during fertilisation in the mouse with possible implications in human reproduction clinics, such as failures in tPA production, which could be partially resolved by the addition of exogenous tPA during IVF treatment.

Physiology learning for veterinary students: impact of guided practices on students' opinion and physiological parameters.

Over recent decades, education has increasingly focused on student-centered learning. Guided practices represent a new way of learning for undergraduate students of physiology, whereby the students turn into teacher-students and become more deeply involved in the subject by preparing and teaching a practical (laboratory) class to their peers. The goal was to assess the students' opinions about guided practices and how physiological parameters change during the activity. For this objective, two experiments were performed. First, a voluntary questionnaire on guided practices was completed by the students during 2 academic years. Students could also write a free text commentary. The positive answers obtained in the questionnaire and the free commentary responses point to the effectiveness of this methodology in students' minds. Negative aspects included the time spent preparing the activity, and the stress that students experienced in the teaching role. Second, information about how the teacher-students felt before teaching the practical class was self-reported, and physiological parameters related to stress (heart rate, pulse rate, blood pressure, arterial oxygen saturation, respiratory rate, and electrocardiogram recorded to evaluate R-R interval and heart rate variability) were measured immediately before and while the practical class was taught. This evaluation reported an increase in stress during the execution of the practice. In conclusion, despite a new and stressful situation, guided practices are of interest for the students as a learning tool and for the acquisition of skills that may be of use in their later professional lives.

Optimization of post-cervical artificial insemination in gilts: Effect of cervical relaxation procedures and catheter type.

Post-cervical (pC) artificial insemination (AI) has been successfully developed for application in multiparous sows, although it has proved problematic in gilts. This study analyzes the use of pC-AI in gilts by two experiments. In the first experiment, the efficiency of pC-AI in gilts was evaluated using a multi-ring multiparous catheter (MpC), which led to 23.1% of the gilts being successfully inseminated. In gilts where insemination was not possible using an MpC, two alternatives were applied before a second attempt at insemination: 1) Vetrabutin Chlorhydrate (VC) was intramuscularly injected in order to relax the cervix; or 2) Warm extender (WE) was deposited in the cervix to modify the cervical muscle dynamics. After the application of these treatments, the success rates achieved with the MpC were 34.2% and 23.8% for VC and WE, respectively. No statistically significant differences were found in the reproductive parameters measured [farrowing (%), litter size and fecundity index] between the use of MpC, or the MpC combined with VC or WE, compared with gilts inseminated by cervical AI (control group). In the second experiment, new catheters based on the anatomical characteristics of gilts (GpC) were used, and the rate of successful pC-AI application were compared (experiment 2a): a) MpC: control; b) GpC1: multi-ring catheter of Ø 16 mm and inner cannula of Ø 3.5 mm; c) GpC2: a multi-ring catheter of Ø with an inner cannula of Ø 2.5 mm. The highest rate of successful cannula penetration was reached in the GpC2 group (60.3%) followed by GpC1 (37.0%) and MpC (19.6%) (p < 0.05). There were no differences in the above mentioned reproductive parameters using the three catheters compared with cervical AI method (control group). Moreover, prior cervical AI did not improve subsequent pC-AI application 24 h later (experiment 2b). In conclusion, Vetrabutin Chlorhydrate, warm extender or the new catheters can be considered as useful tools for improving the success rate of pC-AI technique in gilts.

DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids.

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

Oviductal epithelial cells selected boar sperm according to their functional characteristics.

The interaction of oviductal epithelial cells (OECs) with the spermatozoa has beneficial effects on the sperm functions. The aim of this study is to evaluate the in vitro fertilizing capacity of incubating spermatozoa previously selected by density gradient in OEC and determinate some sperm characteristics that could explain the results obtained. In this study, we assessed in vitro fertilization (IVF), tyrosine phosphorylation, phosphatidylserine translocation, nuclear DNA fragmentation, and chromatin decondensation. Three experimental sperm groups, previously selected by Percoll gradient, were established according to the origin of the sperm used for IVF: (i) W30 group: spermatozoa were incubated with oocytes in the absence of OEC; (ii) NB group: after sperm incubation in OEC, the unbound spermatozoa were incubated with oocytes, in the absence of OEC; and (iii) B group: after sperm incubation with OEC, the bound spermatozoa were incubated with oocytes in the OEC plates. The results showed that sperm from the NB group led to a lower IVF yield, accompanied by low penetration rates (NB: 19.6%, B: 94.9%, and W30: 62.9%; P < 0.001) and problems of nuclear decondensation. Moreover, higher levels of tyrosine phosphorylation were observed in the NB group compared with the W30 and B groups (NB: 58.7%, B: 2.5%, and W30: 4.5%; P < 0.01). A similar trend was observed in phosphatidylserine translocation (NB: 93.7%, B: 5.7%, and W30: 44.2%; P < 0.01). These results demonstrate that the OEC exerts a rigorous degree of sperm selection, even within an already highly selected population of spermatozoa, and can capture the best functional spermatozoa for fertilization.

Importance of sperm morphology during sperm transport and fertilization in mammals.

After natural or artificial insemination, the spermatozoon starts a journey from the site of deposition to the place of fertilization. However, only a small subset of the spermatozoa deposited achieves their goal: to reach and fertilize the egg. Factors involved in controlling sperm transport and fertilization include the female reproductive tract environment, cell-cell interactions, gene expression, and phenotypic sperm traits. Some of the significant determinants of fertilization are known (i.e., motility or DNA status), but many sperm traits are still indecipherable. One example is the influence of sperm dimensions and shape upon transport within the female genital tract towards the oocyte. Biophysical associations between sperm size and motility may influence the progression of spermatozoa through the female reproductive tract, but uncertainties remain concerning how sperm morphology influences the fertilization process, and whether only the sperm dimensions per se are involved. Moreover, such explanations do not allow the possibility that the female tract is capable of distinguishing fertile spermatozoa on the basis of their morphology, as seems to be the case with biochemical, molecular, and genetic properties. This review focuses on the influence of sperm size and shape in evolution and their putative role in sperm transport and selection within the uterus and the ability to fertilize the oocyte.

Oviductal Transcriptome Is Modified after Insemination during Spontaneous Ovulation in the Sow.

Gene Expression Microarray technology was used to compare oviduct transcriptome between inseminated and non-inseminated pigs during spontaneous oestrus. We used an in vivo model approaching the study from a physiological point of view in which no hormonal treatment (animals were in natural oestrus) and no artificial sperm selection (selection was performed within the female genital) were imposed. It is therefore emphasised that no surgical introduction of spermatozoa and no insemination at a site other than the physiological one were used. This approach revealed 17 genes that were two-fold or more up-regulated in oviducts exposed to spermatozoa and/or developing embryos and 9 genes that were two-fold or more down-regulated. Functional analysis of the genes revealed that the top canonical pathways affected by insemination were related to the inflammatory response and immune system (Network 1) to molecular transport, protein trafficking and developmental disorder (Network 2) and to cell-to-cell signalling and interaction (Network 3). Some of the genes in network 1 had been previously detected in the oviduct of human and animals, where they were over-expressed in the presence of spermatozoa or pre-implantation embryos (C3, IGHG1, ITIH4, TNF and SERPINE1) whereas others were not previously reported (SAA2, ALOX12, CD1D and SPP1). Genes in Network 2 included RAB1B and TOR3A, the latter being described for the first time in the oviduct and clearly expressed in the epithelial cells of the mucosa layer. Network 3 integrated the genes with the highest down-regulation level (CYP51, PTH1R and TMOD3). Data in the present study indicate a change in gene expression during gamete encounter at the site of fertilization after a natural sperm selection within the female genital tract. These changes would indicate a modification of the environment preparing the oviduct for a successful fertilization and for an adequate embryo early development.

Biphasic role of calcium in mouse sperm capacitation signaling pathways.

Mammalian sperm acquire fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with up-regulation of a cAMP-dependent pathway, changes in intracellular pH, intracellular Ca(2+), and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca(2+) ions have a biphasic role in the regulation of cAMP-dependent signaling. Media without added Ca(2+) salts (nominal zero Ca(2+)) still contain micromolar concentrations of this ion. Sperm incubated in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca(2+). However, chelation of the extracellular Ca(2+) traces by EGTA induced both cAMP-dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca(2+) media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca(2+) ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin. Interestingly, contrary to wild-type mouse sperm, sperm from CatSper1 KO mice underwent PKA activation and an increase in tyrosine phosphorylation upon incubation in nominal zero Ca(2+) media. Therefore, sperm lacking Catsper Ca(2+) channels behave as wild-type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca(2+) involved in the regulation of cAMP-dependent and tyrosine phosphorylation pathways required for sperm capacitation.

Reproductive performance and backflow study in cervical and post-cervical artificial insemination in sows.

The present study was developed to evaluate multiparous sow reproductive performance and backflow in post-cervical artificial insemination (post-CAI) using a reduced number of sperm than in cervical artificial insemination (CAI). The experimental groups were divided into sows inseminated by: 1) cervical artificial insemination (CAI): 3×10(9) spermatozoa/80 ml; 2) post-CAI: 1.5×10(9) spermatozoa/40 ml (post-CAI 1); 3) post-CAI using 1×10(9) spermatozoa/26 ml (post-CAI 2). Post-CAI 1 reproductive parameters were similar to those of post-CAI 2 (except for live born litter size which was greater in post-CAI 1) and better than for the CAI group (p<0.01). In a second experiment the backflow volume, number of sperm, and sperm quality in the backflow were studied in the 3 experimental groups. The % of volume and spermatozoa in the backflow was higher in the CAI group (p<0.05) than post-CAI groups (statistically similar between them). Moreover, the quality parameters (motility, progressive motility, viability, chromatin decondensation and morphology) in backflow semen were identical in all three experimental groups, but differed as regards the original insemination dose incubated inside a colostomy bag (sperm quality control group). The present study shows that the use of post-CAI (either post-CAI 1 or 2) in field conditions can be recommended because the efficiency is similar (in the case of post-CAI 2) or higher (post-CAI 1) than when using the traditional method (CAI), representing a reduction cost.