L. Vieira

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.

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.

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.

Equine spermatozoa stored in the epididymis for up to 96 h 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.

Role of insulin-like growth factor-I and follicular fluid from ovarian follicles with different diameters on porcine oocyte maturation and fertilization in vitro

The objective of this study was to determine the effects of insulin-like growth factor-I (IGF-I) (0, 60, 120, 180, and 240 ng/mL) and follicular fluid (FF) derived from 2 to 5 and 6 to 10 mm diameter follicles (SpFFs and LpFFs, respectively) added during in vitro maturation (IVM) of porcine oocytes on nuclear maturation and IVF. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium supplemented with SpFFs or LpFFs and various IGF-I concentrations. The COCs were cultured for 44 hours, and then fertilized in vitro. Maturation and IVF results were recorded 18 hours after insemination. The IVM (%) was higher (P < 0.05) in the COCs matured in LpFFs than with SpFFs when 0 (90.0 ± 6.9 vs. 76.3 ± 10.7) or 60 ng/mL IGF-I (92.0 ± 8.1 vs. 81.8 ± 10.2) was added. In SpFFs media, there was a quadratic relationship (P < 0.01) between IGF-I concentration and IVM (peak results at IGF-I = 129 ng/mL). However, when the COCs were matured with LpFFs, there was a decreasing linear effect between IGF-I concentration and IVM. At all concentrations of IGF-I, the percentage of degenerated oocytes was higher in COCs matured in SpFFs than in LpFFs. Penetration (%) did not differ (P > 0.05) between COCs matured with SpFFs or LpFFs when 60 (66.8 ± 9.4 vs. 72.7 ± 11.3) or 180 ng/mL of IGF-I (75.7 ± 10.4 vs. 73.8 ± 13.2) were used. Monospermy (%) was similar between SpFFs and LpFFs only with addition of 120 ng/mL IGF-I. The IVF performance (%) did not differ between COCs matured with SpFFs or LpFFs when IGF-I concentrations of 120 (28.5 ± 8.8 vs. 38.5 ± 8.3) and 180 ng/mL (24.3 ± 10.2 vs. 30.12 ± 8.2) were used. There was no effect of IGF-I concentration or of FF type on the number of penetrated sperm per oocyte and on male pronuclear formation. For COCs matured with SpFFs, there was a quadratic relationship between IGF-I concentration and penetration, monospermy, and IVF performance (peak results at IGF-I = 179, 122, and 135 ng/mL, respectively). Thus, on the basis of the observed quadratic relationships, we inferred that when using SpFFs, the addition of IGF-I (122-179 ng/mL) to the IVM medium produced results similar to those obtained with LpFFs without adding IGF-I. In conclusion, the addition of IGF-I to the IVM medium supplemented with SpFFs increased maturation and improved IVF results. Alternatively, IGF-I had no effect on IVM or IVF when used with LpFFs.

In vitro fertilization of porcine oocytes is affected by spermatic coincubation time

The aim was to study the effects of different gamete coincubation times on porcine in vitro fertilization (IVF), and to verify whether efficiency could be improved by reducing oocyte exposure time to spermatozoa during IVF. In groups of 50, a total of 508 immature cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium. The COCs were cultured for 44 hours and then inseminated with in natura semen (2,000 spermatozoa/oocyte). The sperm and oocytes were coincubated according to the following treatments (T): T1 = oocytes exposed to spermatozoa for one hour (173 oocytes), T2 = oocytes exposed to spermatozoa for two hours (170 oocytes), and T3 = oocytes exposed to spermatozoa for three hours (165 oocytes). After these coincubation periods, the oocytes were washed in fertilization medium (TALP medium) to remove spermatozoa not bound to the zona pellucida and cultured in another similar medium (containing no sperm). Eighteen to twenty hours after fertilization, the putative zygotes were stained in Hoechst-33342 to evaluate the IVF results. The penetration rate was higher (P 0.05) between oocytes exposed to spermatozoa for one (T1) and three hours (T3). However, optimum (P=0.048) results were obtained after two hours of coincubation, when the rate of fertilization performance was 50.16±8.52%. The number of penetrated sperm per oocyte, as well as male pronucleus formation, did not differ (P>0.05) between the treatments evaluated. Under these assay conditions, especially in relation to the sperm concentration used, gamete coincubation for a period of two hours appears to be optimal for monospermy and fertilization performance. Thus, it is the optimal time period for obtaining a large number of pig embryos capable of normal development.

Evaluación de la viabilidad y motilidad espermática dosis seminales caprinas para el desarrollo de un modelo de contaminación experimental con mycoplasma spp

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