Joaquin Gadea

Prediction of porcine semen fertility by homologous in vitro penetration (hIVP) assay.

A field trial was conducted to compare the fertility predicting capacity of different sperm assays applying classical semen analysis, sperm function and the homologous in vitro penetration test (hIVP) to 60 ejaculates from four boars collected over a period of 15 weeks. No differences were found between the groups of fertility (Low Fertility: < 20%; Intermediate: 40-60% and High: > 80%) for sperm-rich fraction volume collection, sperm concentration, total sperm number, cationic contents in seminal plasma and ATP concentration. Partial differences were found in the parameters of motility, normal morphology, normal apical ridge (NAR), viability with eosin-nigrosin stain, hypo-osmotic swelling test (HOS), osmotic resistance test (ORT) and functional membrane integrity (with carboxyfluorescein diacetate, DCF). These parameters would be useful for detecting sperm with poor fertility, but they are not precise enough to discriminate an ejaculate with higher fertility than the herd median. Only the penetration percentage (10.24 +/- 1.45 vs. 55.13 +/- 3.35 vs. 84.72 +/- 1.73) and sperm number per oocyte (1.29 +/- 0.07 vs. 11.29 +/- 1.79 vs. 25.86 +/- 1.43) in a hIVP system were parameters with a predictive capacity to discriminate between the three fertility groups. Consequently, hIVP was found to be the best seminal assay and it may improve the in vitro assessment of sperm fertilizing ability.

Evaluation of boar spermatozoa penetrating capacity using pig oocytes at the germinal vesicle stage

We evaluated the ability of immature pig oocytes (at germinal vesicle stage) to detect differences in the in vitro penetration rates of boar spermatozoa. In Experiment 1, immature and ovulated oocytes (n=303) were exposed to capacitated boar spermatozoa to determine if the penetrability of immature pig oocytes was comparable to that of ovulated oocytes. The percentages of penetrated oocytes and the mean number of spermatozoa per oocyte were similar for immature (88.82 and 7.42+/-0.41) and ovulated oocytes (90.97 and 7.95+/-0.34, respectively). In Experiment 2, immature oocytes (n=1230) were inseminated with semen from 2 boars (A and B) with satisfactory semen characteristics to establish the variability of in vitro penetrating capacity between the boars. Semen was examined for motility, movement quality, acrosome integrity and plasma membrane integrity at various stages of the in vitro procedure. Although the sperm evaluation results were similar between boars, Boar A exhibited a significantly higher (P<0.001) penetration rate (91.49%) and number of spermatozoa penetrated per oocyte (5.90+/-0.25) than Boar B (52.87% and 2.03+/-0.12, respectively). Increasing the sperm concentration at insemination from 1 x 10(6) to 10 x 10(6) cells/ml resulted in an increased penetrating capacity for both boars, and the differences in the number of spermatozoa per oocyte between boars also increased. These results indicate that immature pig oocytes can be used in a homologous in vitro fertilization assay, and that despite similarities in semen characteristics a significant boar effect is evident for parameters of in vitro penetration of oocytes.

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 factors related to in vitro penetration of porcine oocytes.

This study was designed to evaluate the relationship between sperm factors and penetration capacity in an in vitro system with immature porcine oocytes. The sperm parameters evaluated in 145 ejaculates were volume, sperm concentration, total cells in the ejaculate, ATP content, morpho-anomalies, percentage of motile sperm cells, forward progressive motility (FPM), acrosome status (NAR), hypo-osmotic swelling test (HOS), osmotic resistance test (ORT), eosin-nigrosin viability stain and sperm membrane integrity (DCF). Porcine oocytes (a total of 8,736) were used to evaluate the capacity of the different sperm assays to predict penetration. Many parameters were found to be related to in vitro penetration ability; all conventional semen parameters, except sperm concentration and eosinnigrosin staining, were significantly better in high (>75%) than in low penetration rates (<75%). When the ejaculates were preselected the number of significantly related parameters was lower. When studying all conventional semen parameters through a stepwise multiple linear regression analysis of seminal measurements, up to 72.3% of total variance of the penetration rate could be predicted. However, as many as 4 parameters were needed (FPM in fresh semen, folded tail, NAR in post-treatment semen and DCF) for accurate prediction. On the other hand, the multiple logistic regression needed 7 parameters to discriminate 83.96% of the cases correctly. In summary, the results from the present study showed that almost all studied parameters were significantly different for predicting penetration process attained or failed, but most of them were correlated together. These findings emphasize the complexity of sperm functions and the difficulty of assessing the fertilizing ability.

Determination of glycosidase activity in porcine oviductal fluid at the different phases of the estrous cycle.

Sperm-oocyte binding and gamete-oviductal epithelium interactions are carbohydrate-mediated events occurring in the oviductal fluid (OF). Thus, knowledge about the activities of glycosidases (enzymes catalyzing hydrolytic cleavage of terminal sugar residues) in this milieu would help us understand the molecular mechanisms involved in these events. This work was carried out to investigate the glycosidase activity, protein content, and volume of OF collected from gilts and sows. Oviducts were classified into four phases of the estrous cycle (early follicular, late follicular, early luteal, and late luteal) based on the appearance of the ovaries. OF was aspirated, centrifuged, measured for volume, and frozen until assay. Substrates conjugated to 4-methylumbelliferyl were used to screen the activities of seven different glycosidases at physiological pH (7.2). alpha-L-Fucosidase and beta-N-acetyl-glucosaminidase activities increased at the late follicular phase to decrease after ovulation. beta-D-Galactosidase, alpha-D-mannosidase, and beta-N-acetyl-galactosaminidase showed higher activities at the early follicular phase, which decreased after ovulation. N-Acetyl-neuraminidase and alpha-D-galactosidase did not show activity at any phase of estrous cycle neither in sows nor in gilts at pH 7.2, although it did at acidic pH (4.4) in the follicular and luteal phase samples. Total protein also changed during the cycle showing the maximum secretion at the late follicular phase (2118.6+/-200.7 microg/oviduct). The highest volumes of OF were collected from the oviducts at the late follicular phase (50.7+/-1.3 microl/oviduct). These results indicate that OF from sows and gilts shows glycosidase activity varying throughout the estrous cycle suggesting a role of these enzymes in carbohydrate-mediated events.

Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro fertilizing ability of frozen‐thawed bull spermatozoa

In this study, we evaluated the effects of glutathione (l-gamma-glutamyl-l-cysteinylglycine; GSH) supplementation of the thawing extender on bull semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To address these questions fully, we used a set of functional sperm tests. These included tests of sperm motility assayed by computer-assisted semen analysis, membrane lipid packing disorder, spontaneous acrosome reaction, free radical production [reactive oxygen species (ROS) generation], sperm chromatin condensation, DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling and acridine orange staining measured by flow cytometry. Finally, the in vitro penetrability of in vitro matured oocytes and the in vitro production of embryos were evaluated. The main findings emerging from this study were that addition of GSH to the thawing medium resulted in: (i) a higher number of non-capacitated viable spermatozoa; (ii) a reduction in ROS generation; (iii) lower chromatin condensation; (iv) lower DNA fragmentation; (v) higher oocyte penetration rate in vitro and (vi) higher in vitro embryo production compared with control group. Nevertheless, GSH had no significant effect on motion parameters or the occurrence of the spontaneous acrosome reaction. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen bull spermatozoa.

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 spermatozoas 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.

In vitro penetration assay of boar sperm fertility: Effect of various factors on the penetrability of immature pig oocytes

The present study was designed 1) to examine the influence of cumulus cells, ovary storage time and oocyte size on the penetrability of immature pig oocytes, and 2) to investigate the effect of 2 methods of treating the semen from different boars on the inter-assay variability of homologous in vitro penetration tests of boar sperm fertility. In Experiment 1, cumulus oocyte complexes, oocytes with spontaneous loss of the cumulus cells during collection, and oocytes mechanically stripped of cumulus cells were used. No differences were observed in oocyte penetrability among the 3 types of oocyte, although mechanical removal of the cumulus caused an increase (P < 0.005) in the degeneration rate compared with the other oocyte types. In Experiment 2, the oocytes were recovered from ovaries kept in PBS (30 degrees C) for 2, 4 or 6 h after slaughter of prepuberal gilts. Ovary storage did not modify the penetrability of oocytes but increased (P < 0.02) their degeneration rates. In Experiment 3, the diameters of fresh oocytes were determined after co-incubation with spermatozoa. They were classified into 4 groups according to diameter: A) < 105 microm, B) 105-115 microm, C) 116-120 microm and D) > 120 microm. Oocytes from Groups C and D exhibited higher (P < 0.05) penetrability than oocytes from the other groups. In Experiment 4, stored, diluted spermatozoa from 4 boars were pretreated by centrifugation at 50 x g for 3 min and subsequent concentration of the supernatants at 1,200 x g for 3 min. The pellets were treated (washed twice and preincubated for 40 minutes) before co-incubation with immature oocytes or used directly as untreated samples (unwashed and non-preincubated). A boar effect (P < 0.001) was evident for the parameters of in vitro penetration, independently of sperm treatment. When the oocytes were inseminated with untreated spermatozoa, the effects of the replicate and the boar-by-replicate interaction on the variability in oocyte penetrability were not significant. The results of this study indicate that the use of standardized immature pig oocytes and stored untreated, diluted spermatozoa can provide a useful method for optimizing the homologous in vitro penetration (hIVP) assay of boar fertility.

Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA-DNA complexes at 5 microg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT-ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

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 Dulbeccos 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.