Boar sperm motility is modulated by CCK at a low concentration of bicarbonate under capacitation conditions.

In a previous study, our group detected the cholecystokinin (CCK) protein in the porcine oviduct. This fact, together with the involvement of CCK in the regulation of sperm protein tyrosine phosphorylation by the modulation of HCO3 - uptake (in mice and humans) suggests a role for CCK during sperm capacitation. Therefore, on the one hand, the expression of CCK receptors (CCK1R and CCK2R) on boar testes has been investigated and probed; on the other hand, boar spermatozoa (from seminal doses of 1-day and 5-day storage) were exposed to different concentrations of CCK (0-control, 25 or 50 µM) in a medium supporting capacitation supplemented with 0, 5 or 25 mmol/L of HCO3 - for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 µM of CCK) were observed when HCO3 - was absent in the media (p > .05). However, the results showed that when the media was supplemented with 5 mmol/L HCO3 - in 1-day seminal dose storage, the linearity index (LIN, %), straightness index (STR, %) and oscillation index (WOB, %) (sperm kinetics parameters) increased in the presence of CCK regardless the concentration (p < .05). Nevertheless, CCK in sperm from 5-day storage only increased the WOB parameter in comparison to the control (p < .05). Furthermore, the average amplitude of the lateral displacement of the sperm head (ALH, µm) and curvilinear velocity (VCL, µm/s) decreased when CCK was present, depending on its concentration and sperm aging (1-day vs. 5-days) (p < .05). In the case of the media supporting capacitation supplemented with 25 mmol/L HCO3 - , any differences were observed except for sperm viability in the 5-day seminal doses, which increased in the 50 µM-CCK group compared to the control (p < .05). In conclusion, these data suggest an implication of CCK protein during sperm capacitation under low bicarbonate concentration increasing the sperm linear trajectory.

In vitro supplementation of testosterone or prolactin affects spermatozoa freezability in small ruminants.

In small ruminants, testosterone and prolactin plasma concentrations show circannual fluctuations as an adaptation mechanism to their seasonal breeding behavior. Sperm resistance to the freezing-thawing process shows seasonal fluctuation throughout the year, with lower sperm freezability at the beginning of the breeding season when prolactin and testosterone levels reach their maximum concentration. Nevertheless, whether these hormones directly affect post-thaw sperm quality parameters is still unclear. The objective was to study the effect of testosterone or prolactin added in vitro on sperm freezability in domestic ram (Ovis aries) and buck (Capra hircus). Sperm samples were incubated for 1 h with a range of testosterone (0, 2, 4, or 6 ng/mL; Exp. 1) or prolactin (0, 20, 100, 200, or 400 ng/mL; Exp. 2) concentrations. Samples were cryopreserved by slow freezing in straws at 0 h and after 1 h incubation. Sperm viability, acrosome integrity, motility, and kinetic parameters were assessed at 0 and 1 h in fresh and frozen-thawed samples. Results showed no hormone effect in fresh sperm, whereas these hormones affected post-thaw sperm parameters. In Exp. 1, in vitro incubation with testosterone decreased the post-thaw acrosome integrity of ram sperm (from 68.1 ± 6.3% to 49.6 ± 3.9%; P < 0.05). In Exp. 2, in vitro incubation with prolactin decreased the post-thaw acrosome integrity of ram (from 78.2 ± 3.4% to 66.3 ± 3.5%; P < 0.05) and buck sperm (from 81.7 ± 2.5% to 67.6 ± 3.5%; P < 0.05). Moreover, prolactin increased the post-thaw amplitude of lateral head displacement in ram sperm (from 3.3 ± 0.1 µm to 3.8 ± 0.2 µm; P < 0.05). In conclusion, either testosterone or prolactin added in vitro decreased the post-thaw acrosome integrity of ram and buck sperm. This suggests a destabilization process that could be decreasing sperm freezability when physiological levels of these hormones are high in vivo.

Effect of boar seminal dose type (cervical compared with post-cervical insemination) on cooling curve, sperm quality and storage time.

Seminal doses used for cervical and post-cervical artificial insemination (CAI and PCAI, respectively) vary in volume, the number of spermatozoa and packaging. The aim was to evaluate the outcomes when there was use of routine processing procedures for CAI- and PCAI-doses. Two different types of seminal doses were processed: 1) CAI: 2.7 × 109 sperm/80 ml; 2) PCAI: 1.5 × 109 sperm/45 ml. In Experiment 1, the cooling curve of seminal doses during processing occurred in two phases: 1st) At room temperature (23.4 ±â€¯0.5 °C) from 0 (just after packaging) to 120 min; 2nd) At refrigeration (15.7 ±â€¯0.8 °C) from 121-240 min. For the PCAI-doses, the time required to reach room temperature was 47 min compared to 107 min for CAI-doses (decreasing velocity of 0.093 °C/min and 0.048 °C/min, respectively). During refrigeration, for the PCAI-doses the time required to reach the desired preservation temperature was 20 min less than for CAI-doses (PCAI: 90 min, 0.074 °C/min; CAI: 110 min, 0.066 °C/min). In Experiment 2, sperm motility, kinetic parameters and acrosome damage for both types of doses were evaluated at 0, 24, 48 and 72 h of refrigeration. Also, morphology, pH, and osmolality were assessed at 0 and 72 h. Values for all these did not differ between CAI- and PCAI-doses. In conclusion, PCAI-doses took less time than CAI-doses to reach the desired temperature, but sperm quality was similar for CAI- and PCAI-doses during storage. Nevertheless, the different cooling curves should be taken into consideration for further investigation.

Effect of temperature and cell concentration on dolphin (Tursiops truncatus) spermatozoa quality evaluated at different days of refrigeration.

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Sperm cryopreservation in the bottlenose dolphin (Tursiops truncatus) has been developed for the exchange of gametes within the ex situ population. The aim of this study was to develop an effective method for refrigeration of bottlenose dolphin spermatozoa diluted in a commercial extender (BTS). In Experiment 1, the effect of temperature (5 compared with 15 °C) on sperm quality was evaluated during 7 days of storage at 100 × 106 spermatozoa/ml. In Experiment 2, the effect of the storage concentration (100 × 106 compared with 20 × 106 spermatozoa/ml) on sperm quality was assessed during 7 days of storage at 5 °C. In Experiment 1, total motility (including % of rapid sperm) was greater at 5 than 15 °C. When the effect of storage concentration was evaluated (Experiment 2), total motility and ALH were greater at the higher storage concentration (100 × 106 spermatozoa/ml). For both experiments, values for viability, acrosome integrity, and normal morphology variables were consistent throughout the 7 days of refrigeration. In Experiment 3, a microbiological study was performed to evaluate the effect of the refrigeration temperature and days of storage on bacterial growth. The results of microbiological analysis indicated there was Staphylococcus aureus in some samples, however, there was no effect of temperature or days of refrigeration. In conclusion, bottlenose dolphin semen can be refrigerated for a short to medium period of storage and there is maintenance of functionality of sperm when stored at 100 × 106 spermatozoa/ml at 5 °C.

Effect of Porcine circovirus 2 (PCV-2) maternally derived antibodies on performance and PCV-2 viremia in vaccinated piglets under field conditions.

BACKGROUND: Nowadays, the most common presentation of PCV-2 is the subclinical infection in piglets after weaning. The success of PCV-2 vaccination is associated with the control of the clinical disease as well as the improvement of production parameters. In consequence, the objective of the present study was to analyse the effect of PCV-2 maternally derived antibody (MDA) levels on vaccine efficacy in piglets vaccinated at three weeks of age with a commercial PCV-2 subunit vaccine. The study was performed analysing a database with 6112 wean-to-slaughter piglets from 4 different European regions. RESULTS: Results showed that the use of the vaccine was able to decrease the PCV-2 viremia calculated as area under the curve (AUC = 60.29 ± 3.73), increase average daily weight gain (ADWG = 0.65 ± 0.01 kg/day) and reduce mortality (7%) in vaccinated piglets compared to non-vaccinated ones (AUC of 198.27 ± 6.14, 0.62 ± 0.01 kg/day and 11% respectively). The overall difference of ADWG between both groups was close to 30 g per day (p < 0.05), also when they were split for low and high levels of MDA titres. Moreover, the animals with the highest ADWG were observed in the group of piglets vaccinated with high or extremely high antibody titres (0.66 and 0.65 kg/day respectively). Considering only animals with extremely high antibody titres, both study groups performed similar, however there was a numerical difference of 10 g/day in favour of vaccinated piglets. Likewise, lack of correlation between ADWG and MDA was observed suggesting that no maternal antibody interference was present with the tested vaccine because the vaccinated animals grew faster compared to unvaccinated control animals, regardless of the level of maternal antibodies present at the time of vaccination. CONCLUSIONS: The results of the present study demonstrated that the MDA against PCV-2 transferred through the colostrum intake has a protective effect against this viral infection. The vaccine used in the present study (Ingelvac CircoFLEX®) was effective when applied at three weeks of age and was not affected by the level of MDA at the time of vaccination.

Seminal plasma mitigates the adverse effect of uterine fluid on boar spermatozoa.

After natural or artificial insemination, spermatozoa start their journey within the uterus to reach the site of fertilization, but only few of them attain this goal. Part of this spermatozoa loss happens in the uterus, in which uterine fluid (UF) seems to be involved. It is known from other species that UF provokes damage to spermatozoa, which is avoided when seminal plasma (SP) is present. Therefore, the aim of the present study was to evaluate the effect of UF on the quality of ejaculated (previously contacted with SP) and epididymal (without previous contact with SP) boar spermatozoa analyzing motility, kinetic parameters, viability and acrosome integrity in the presence or absence of SP over time. Three experimental groups were evaluated in each source of spermatozoa (ejaculated and epididymal): 1) Control: spermatozoa with 20% of SP; 2) UF: spermatozoa with 20% of UF; and 3) UF-SP: spermatozoa with 20% of SP and 20% of UF. Total and progressive motility, kinetic parameters (VCL, VSL, VAP, LIN, STR, WOB, and BCF), viability and acrosome damage were analyzed at 15, 60, 120 and 180 min after incubation. Total and progressive motility decreased when ejaculated spermatozoa were incubated in UF in contrast to control and UF-SP groups (p < 0.0007), with no differences between control and UF-SP. The VCL decreased in the UF group compared to the control and UF-SP groups in ejaculated spermatozoa (p = 0.0002). The VSL, VAP, LIN and STR kinetic parameters were greater when ejaculated spermatozoa were incubated in the UF-SP group than in the UF group (all: p ≤ 0.02). Acrosome damage increased in ejaculated and epididymal spermatozoa incubated in the UF group compared to the control and UF-SP groups (both: p < 0.0001). Also, the viability of epididymal spermatozoa decreased in the UF group, while it did not change in the control and UF-SP groups (p = 0.0004). The rest of the parameters in either ejaculated or epididymal spermatozoa did not differ between experimental groups, except for WOB when epididymal spermatozoa were used (UF-SP higher than the control group, with UF being similar for both; p = 0.03). In conclusion, both ejaculated and epididymal spermatozoa are affected by UF, suggesting a negative effect on their quality. This negative effect is reduced by the presence of SP, improving the spermatozoa functionality, preserving motility, viability and acrosome integrity.

Post-cervical artificial insemination in porcine: The technique that came to stay.

The porcine industry is of great importance worldwide, and so any technological innovation in one or more of the associated production areas is of interest for meat production. Among such innovations in the reproduction area, post-cervical or intrauterine artificial insemination (PCAI) has emerged as a new approach in artificial insemination (AI). PCAI is gradually replacing traditional cervical insemination (CAI), particularly in countries with intensive pig production industries. This type of insemination, which deposits the semen in the body of the uterus (as opposed to traditional cervical deposition), is increasingly used in the field due to its simplicity and the numerous advantages that it provides at production level (e.g. reduced number of sperm, less time required to perform insemination and faster genetic improvement) and, consequently, from an economic point of view. In addition, since its inception, PCAI has been combined with other reproductive biotechnologies, such as the use of frozen-thawed sperm, fixed-time AI or sperm-mediated gene transfer. However, despite its wide acceptance and application, new approaches for increasing the efficiency of PCAI are constantly being sought, such as the adjustment and standardization in sperm numbers, the conservation of the PCAI semen dose, its association with other biotechnologies (sex-sorted sperm) or its efficacy in young (nulliparous and primiparous) females.

Morphological changes in the porcine cervix: A comparison between nulliparous and multiparous sows with regard to post-cervical artificial insemination.

In recent decades, new artificial insemination (AI) methods, such as post-cervical AI (PCAI), have been developed in pig. PCAI involves crossing the cervix to deposit the sperm in the uterine body. Although PCAI application in sows is frequent, its application in nulliparous (gilts) females it is still limited due to the difficulty of passing through the cranial part of the cervical lumen. We hypothesized that ageing and parity would modify the cervical canal, facilitating the introduction of AI devices through the cervix. The aim was to compare the morphology of the uterus at different levels between multiparous and nulliparous females. Morphological analysis of the uterus pointed to a longer cervix (25.9 ±â€¯4.6 vs. 21.6 ±â€¯3.3 cm, p < 0.001) and greater length of the part of the reproductive tract involved in PCAI (from rima vulvae to the last cervical cushion) (56.2 ±â€¯6.0 vs. 50.3 ±â€¯5.2 cm, p < 0.001) in multiparous sows compared with nulliparous animals. As regards the structure of the vaginal and uterine parts of the cervix (the part in contact with the vagina and uterine body, respectively), the cross-sectional area, perimeter and total thickness were greater in the uterine part of multiparous than of nulliparous animals (area: 4.07 ±â€¯1.46 vs. 2.46 ±â€¯0.56 cm2, p < 0.01; perimeter: 8.50 ±â€¯1.44 cm vs. 6.28 ±â€¯0.92 cm, p < 0.001; thickness: 10.79 ±â€¯0.96 vs. 8.35 ±â€¯0.62 mm, p < 0.05), but not in the vaginal part. The tissue content analysed in histological cross-sections also showed differences between female groups, a greater content of connective tissue (58.86 ±â€¯10.78 vs. 67.60 ±â€¯13.38%, p < 0.001) and a lower amount of muscle fibres (39.79 ±â€¯10.24 vs. 30.66 ±â€¯13.69%, p < 0.001) being observed in multiparous sows. Finally, silicone casts of the cervical lumen revealed differences between the two groups in the size and shape of the ridges in the lumen trajectory. Parity, which is also influenced by ageing, determines important changes in the size, structure and tissue content of the cervix wall, as well as in the morphology of the cervical canal, which may be responsible for the different levels of performance of PCAI in the female populations. Therefore, the future design of AI strategies and catheters should take into consideration the morphological variations of the cervix lumen, which will depend on age and parity of the females.

Four glycoproteins are expressed in the cat zona pellucida.

The mammalian oocyte is surrounded by a matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding and may be involved in speciation. In cat (Felis catus), this matrix is composed of at least three glycoproteins called ZP2, ZP3, and ZP4. However, recent studies have pointed to the presence of a fourth protein in several mammals (rat, human, hamster or rabbit), meaning that a reevaluation of cat ZP is needed. For this reason, the objective of this research was to analyze the protein composition of cat ZP by means of proteomic analysis. Using ZP from ovaries and oocytes, several peptides corresponding to four proteins were detected, yielding a coverage of 33.17%, 71.50%, 50.23%, and 49.64% for ZP1, ZP2, ZP3, and ZP4, respectively. Moreover, the expression of four genes was confirmed by molecular analysis. Using total RNA isolated from cat ovaries, the complementary deoxyribonucleic acids encoding cat ZP were partially amplified by reverse-transcribed polymerase chain reaction. Furthermore, ZP1 was totally amplified for the first time in this species. As far as we are aware, this is the first study that confirms the presence of four proteins in cat ZP.

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.

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.

Roles of the oviduct in mammalian fertilization.

The oviduct or Fallopian tube is the anatomical region where every new life begins in mammalian species. After a long journey, the spermatozoa meet the oocyte in the specific site of the oviduct named ampulla and fertilization takes place. The successful fertilization depends on several biological processes that occur in the oviduct some hours before this rendezvous and affect both gametes. Estrogen and progesterone, released from the ovary, orchestrate a series of changes by genomic and nongenomic pathways in the oviductal epithelium affecting gene expression, proteome, and secretion of its cells into the fluid bathing the oviductal lumen. In addition, new regulatory molecules are being discovered playing important roles in oviductal physiology and fertilization. The present review tries to describe these processes, building a comprehensive map of the physiology of the oviduct, to better understand the importance of this organ in reproduction. With this purpose, gamete transport, sperm and oocyte changes in the oviductal environment, and other interactions between gametes and oviduct are discussed in light of recent publications in the field.

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.

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.

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)

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.