Cristina Soriano-Úbeda

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.

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. DOI: http://dx.doi.org/10.7554/eLife.23670.001

Timing of oviductal fluid collection, steroid concentrations, and sperm preservation method affect porcine in vitro fertilization efficiency

OBJECTIVE To determine optimal conditions for the inclusion of oviductal fluid (OF) in IVF protocols. DESIGN Experimental prospective study. SETTING Mammalian reproduction research laboratory. ANIMAL(S) Oviducts and ovaries from porcine females were collected at a slaughterhouse. A total of 30 oviducts and 1,285 oocytes were used. Boar-ejaculated spermatozoa were also used. INTERVENTION(S) In vitro-matured porcine oocytes were preincubated with OF collected from animals before or after ovulation and later fertilized in vitro. Zona pellucida digestion time in oocytes after preincubation in OF was assessed. Concentrations of E2 and P4 in OF were measured. IVF was performed, including within the culture media the E2 and P4 concentrations found in the preovulatory OF. The effect of preovulatory OF on IVF efficiency was compared between fresh and frozen-thawed spermatozoa. MAIN OUTCOME MEASURE(S) E2 and P4 concentrations in OF; penetration and monospermy rates; number of spermatozoa within the ooplasm and on the zona pellucida after IVF under different experimental conditions; zona pellucida resistance to protease digestion. RESULT(S) Preincubation of oocytes in OF collected before ovulation enhances IVF efficiency in the pig compared with OF collected after ovulation (29.58 ± 3.84 vs. 11.03 ± 2.69). When frozen-thawed spermatozoa are used for the IVF of these OF-treated oocytes, their fertilization ability increases compared with fresh semen. OF collected before and after ovulation shows significantly different concentrations of E2 (99.00 ± 8.72 vs. <10 pg/mL) and P4 (2.53 ± 0.66 vs. 12.27 ± 2.33 ng/mL), respectively. Addition of E2 and P4 at concentrations similar to those in the OF before ovulation partially simulates the effect of the fluid on IVF outcome. CONCLUSION(S) Preincubation of oocytes in OF collected before ovulation is a suitable protocol for increasing the efficiency of IVF with fresh semen in the pig model and could be a useful tool to increase the fertilization ability of frozen-thawed spermatozoa in other species. E2 concentrations in preovulatory OF are higher than those reported in blood serum at the same phase of the estrous cycle.

The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters

OVGP1 is the major non-serum glycoprotein in the oviduct fluid at the time of fertilization and early embryo development. Its activity differs among species. Here, we show that the C-terminal region of recombinant OVGP1 regulates its binding to the extracellular zona pellucida and affects its activity during fertilization. While porcine OVGP1 penetrates two-thirds of the thickness of the zona pellucida, shorter OVGP1 glycoproteins, including rabbit OVGP1, are restricted to the outer one-third of the zona matrix. Deletion of the C-terminal region reduces the ability of the glycoprotein to penetrate through the zona pellucida and prevents OVGP1 endocytosis. This affects the structure of the zona matrix and increases its resistance to protease digestion. However, only full-length porcine OVGP1 is able to increase the efficiency rate of in vitro fertilization. Thus, our findings document that the presence or absence of conserved regions in the C-terminus of OVGP1 modify its association with the zona pellucida that affects matrix structure and renders the zona matrix permissive to sperm penetration and OVGP1 endocytosis into the egg.

Morphological study of boar sperm during their passage through the female genital tract

Once deposited in the female tract, sperm face a series of challenges that must be overcome to ensure the presence of an adequate normal sperm population close to the site of fertilization. Our aim was to evaluate the influence of the uterine milieu on boar sperm morphology. In experiment 1, sperm morphology was evaluated in the backflow (60 min after insemination) and within the uterotubal junction (UTJ) (collected ~24 h after insemination) following intrauterine sperm deposition (n = 6) and compared with the morphology of the sperm in the insemination dose. In experiment 2, the influence of the uterine fluid (UF) on sperm morphological modifications was evaluated. For this purpose, ejaculated (n = 4) and epididymal (n = 4) sperm were in vitro incubated with or without UF for 2 and 24 h. In both experiments, sperm were classified as normal, having a cytoplasmic droplet (proximal or distal) or having tail defects. The results of experiment 1 pointed to an increase in morphologically abnormal sperm collected in the backflow (27.70%) and a reduction of the same in the UTJ (2.12%) compared with the insemination dose (17.75%) (P < 0.05). In experiment 2, incubation of ejaculated sperm with UF did not provoke any morphological modifications; however, when epididymal sperm were incubated with UF, a pronounced increase in the percentage of normal sperm was evident after 24 h compared with the initial dose (from 25.77% to 53.58%, P < 0.05), mainly due to distal cytoplasmatic droplet shedding (53.22 vs. 20.20%). In conclusion, almost all the sperm that colonize the UTJ had a normal morphology, with part of the abnormal sperm having been discarded in the backflow and part selected/modified on their way to the oviduct. UF seems to influence cytoplasmic distal droplet removal, as demonstrated previously in seminal plasma.

Improving porcine in vitro fertilization output by simulating the oviductal environment

Differences between the in vitro and in vivo environment in which fertilization occurs seem to play a key role in the low efficiency of porcine in vitro fertilization (IVF). This work proposes an IVF system based on the in vivo oviductal periovulatory environment. The combined use of an IVF medium at the pH found in the oviduct in the periovulatory stage (pHe 8.0), a mixture of oviductal components (cumulus-oocyte complex secretions, follicular fluid and oviductal periovulatory fluid, OFCM) and a device that interposes a physical barrier between gametes (an inverted screw cap of a Falcon tube, S) was compared with the classical system at pHe 7.4, in a 4-well multidish (W) lacking oviduct biological components. The results showed that the new IVF system reduced polyspermy and increased the final efficiency by more than 48%. This higher efficiency seems to be a direct consequence of a reduced sperm motility and lower capacitating status and it could be related to the action of OFCM components over gametes and to the increase in the sperm intracellular pH (pHi) caused by the higher pHe used. In conclusion, a medium at pH 8.0 supplemented with OFCM reduces polyspermy and improves porcine IVF output.