J. Sanchez-Osorio

Early Developing Pig Embryos Mediate Their Own Environment in the Maternal Tract

The maternal tract plays a critical role in the success of early embryonic development providing an optimal environment for establishment and maintenance of pregnancy. Preparation of this environment requires an intimate dialogue between the embryo and her mother. However, many intriguing aspects remain unknown in this unique communication system. To advance our understanding of the process by which a blastocyst is accepted by the endometrium and better address the clinical challenges of infertility and pregnancy failure, it is imperative to decipher this complex molecular dialogue. The objective of the present work is to define the local response of the maternal tract towards the embryo during the earliest stages of pregnancy. We used a novel in vivo experimental model that eliminated genetic variability and individual differences, followed by Affymetrix microarray to identify the signals involved in this embryo-maternal dialogue. Using laparoscopic insemination one oviduct of a sow was inseminated with spermatozoa and the contralateral oviduct was injected with diluent. This model allowed us to obtain samples from the oviduct and the tip of the uterine horn containing either embryos or oocytes from the same sow. Microarray analysis showed that most of the transcripts differentially expressed were down-regulated in the uterine horn in response to blastocysts when compared to oocytes. Many of the transcripts altered in response to the embryo in the uterine horn were related to the immune system. We used an in silico mathematical model to demonstrate the role of the embryo as a modulator of the immune system. This model revealed that relatively modest changes induced by the presence of the embryo could modulate the maternal immune response. These findings suggested that the presence of the embryo might regulate the immune system in the maternal tract to allow the refractory uterus to tolerate the embryo and support its development.

Effect of the cryoprotectant concentration on the in vitro embryo development and cell proliferation of OPS-vitrified porcine blastocysts.

Our objective was to study the effect of the concentration of ethylene glycol (EG) and dimethyl sulfoxide (Me2SO) during vitrification on the development of porcine blastocysts. Vitrification was performed with 0.4 M sucrose and either a Me2SO and EG mixture (15%, 16% and 17% v/v of each) or EG alone (40% v/v), using superfine open pulled straws. Fresh and vitrified blastocysts were cultured for 48 h and the survival and hatching rates were evaluated. Some vitrified and fresh embryos were processed for Hoechst 33342 staining and proliferation cell nuclear antigen (PCNA) inmunolocalization to determine the proliferation index. The survival rate was similar for fresh and vitrified blastocysts, except for blastocysts vitrified using 15% of cryoprotectants, which displayed lower (P < 0.05) survival than fresh blastocysts. Vitrified and fresh blastocysts had a similar cell proliferation index (range: 75.8+/-3.2 to 83.7+/-3). When only hatched blastocysts among groups were compared, the proliferation rate decreased (P < 0.05) after vitrification with 17% of EG-Me2SO. In conclusion, the concentration of EG-Me2SO could be decreased to 16% in the vitrification medium with no reduction of the in vitro developmental ability of the blastocysts. In addition, a 40% EG-based medium can be used for vitrification with similar results to those achieved with a medium containing 16% EG-Me2SO.

Vitrification and warming of in vivo-derived porcine embryos in a chemically defined medium.

The aim of this study was to design a protocol for vitrification and warming of porcine embryos in a chemically defined medium. A total of 663 morulae and blastocysts were collected from weaned crossbred sows (Large White-Landrace) 5 to 6 d after estrus and vitrified with the Superfine Open Pulled Straw method. In Experiment 1, embryos were vitrified using as a basic medium TCM-199-HEPES supplemented with 20% newborn calf serum (NBCS) or with 0, 0.1%, 0.5%, or 1% polyvinyl alcohol (PVA). Nonvitrified embryos were used as a fresh control group. Survival and hatching rates were evaluated after 72 h of in vitro culture to assess embryo viability. In addition, some hatched blastocysts derived from morulae and blastocysts were processed to determine the total cell number and the cell proliferating index as measures of their quality. Within each stage of embryo development, the different vitrification groups and the fresh control group showed similar high embryo survival (range, 70.5+/-7.1% to 84.9+/-8.1% and 85.3+/-8.1% to 98.4+/-8.2% for morulae and blastocysts, respectively) and hatching rate (range, 46.3+/-10.1% to 66.7+/-11.2% and 73.7+/-11.3% to 89.4+/-11.2% for morulae and blastocysts, respectively) and quality after in vitro culture. In Experiment 2, embryos were vitrified using 0.1% PVA and warmed with TCM-199-HEPES-0.13 M sucrose supplemented with 20% NBCS or either 0 or 0.1% PVA. Nonvitrified embryos were used as a fresh control group. As in Experiment 1, no significant differences were detected in embryo survival (range, 67.9+/-6.6% to 74.5+/-6.6% and 91.9+/-7.0% to 99.5+/-6.3% for morulae and blastocysts, respectively) and hatching rate (range, 47.0+/-7.2% to 64.8+/-9.9% and 89.4+/-7.4% to 98.2+/-6.9% for morulae and blastocysts, respectively) and quality among the warming groups or among vitrified and fresh control embryos. In both experiments, the developmental embryo stage influenced the survival and hatching rates, as well as the number of cells (P<0.01), with the blastocyst stage yielding the best results. In conclusion, PVA can be used as a substitute for serum in vitrification and warming solutions without detrimental effects on the in vitro development of in vivo-derived porcine morulae and blastocysts.

Successful Non-Surgical Deep Uterine Transfer of Porcine Morulae after 24 Hour Culture in a Chemically Defined Medium

Excellent fertility and prolificacy have been reported after non-surgical deep uterine transfers of fresh in vivo-derived porcine embryos. Unfortunately, when this technology is used with vitrified embryos, the reproductive performance of recipients is low. For this reason and because the embryos must be stored until they are transferred to the recipient farms, we evaluated the potential application of non-surgical deep uterine transfers with in vivo-derived morulae cultured for 24 h in liquid stage. In Experiment 1, two temperatures (25°C and 37°C) and two media (one fully defined and one semi-defined) were assessed. Morulae cultured in culture medium supplemented with bovine serum albumin and fetal calf serum at 38.5°C in 5% CO2 in air were used as controls. Irrespective of medium, the embryo viability after 24 h of culture was negatively affected (P<0.05) at 25°C but not at 37°C compared with the controls. Embryo development was delayed in all experimental groups compared with the control group (P<0.001). Most of the embryos (95.7%) cultured at 37°C achieved the full or expanded blastocyst stage, and unlike the controls, none of them hatched at the end of culture. In Experiment 2, 785 morulae were cultured in the defined medium at 37°C for 24 h, and the resulting blastocysts were transferred to the recipients (n = 24). Uncultured embryos collected at the blastocyst stage (n = 750) were directly transferred to the recipients and used as controls (n = 25). No differences in farrowing rates (91.7% and 92.0%) or litter sizes (9.0±0.6 and 9.4±0.8) were observed between the groups. This study demonstrated, for the first time, that high reproductive performance can be achieved after non-surgical deep uterine transfers with short-term cultured morulae in a defined medium, which opens new possibilities for the sanitary, safe national and international trade of porcine embryos and the commercial use of embryo transfer in pigs.

Relevance of ovarian follicular development to the seasonal impairment of fertility in weaned sows.

A field study was conducted to estimate seasonal differences in follicular development in weaned sows and to evaluate the implication of these differences on seasonal infertility. A total of 110 sows were selected at weaning during winter-spring (WS, n=58) and summer-autumn (SA, n=52). Ovaries were scanned once daily from weaning to the onset of oestrus and twice daily from then until ovulation. Six sows during WS were removed from study for not showing growing follicles at weaning. Oestrus was evaluated twice daily from day 1 after weaning to day 14 post-weaning. One of 52 (1.9%) sows in WS and 9/52 (17.3%) in SA showed no signs of oestrus within 14 days of weaning (P<0.05). The diameters of the follicles at weaning, at the onset of oestrus and just before ovulation were smaller (P<0.01) in SA sows than in WS sows. There were fewer follicles in SA sows than in WS sows just before ovulation (P<0.05). Fifty of 51 (98.0%) sows in WS and 31/43 (72.1%) sows in SA experienced a weaning-to-oestrus interval (WOI) of 3-6 days (P<0.05). Fifty-one of 52 (98.1%) sows in WS and 43/52 (82.7%) sows in SA were inseminated; the percentage of pregnant sows that failed to farrow was lower in WS (1/51, 2.0%) than in SA (5/43, 11.6%; P<0.05). The percentage of farrowed sows was greater in WS (46/51, 90.2%) than in SA (32/43, 74.4%; P<0.05). Sows in WS had on average 1.5 more piglets than sows in SA (P<0.05). Sows with a WOI of 3-6 days had lower rates of pregnancy losses (P<0.05) and higher farrowing percentages (P<0.01) than those with a WOI>6 days, irrespective of season.

Non-surgical deep intrauterine transfer of superfine open pulled straw (SOPS)-vitrified porcine embryos: evaluation of critical steps of the procedure.

Previous trials achieved extremely poor results when using the one-step warming method in a syringe in combination with non-surgical deep intrauterine transfer (NET) of superfine open pulled straw (SOPS)-vitrified embryos. This study aimed to assess the effect of the warming procedure on the in vitro and in vivo development of SOPS-vitrified embryos. The effect of the passage of the vitrified-warmed (VW) embryos through the NET catheter was also evaluated. Groups of 4 to 6 morulae and blastocysts, collected from weaned sows, were SOPS-vitrified in 1 μL of vitrification medium, warmed by the one-step warming method in a dish or in a 1-mL syringe and cultured in vitro for 48 h to evaluate the embryo survival (ES) and hatching rates (HR). Warming in syringe had a deleterious effect (P < 0.05) on the in vitro ES (60.5 ± 10.4%) and HR (39.6 ± 9.5%) of VW embryos in comparison with embryos warmed in a dish (85.4 ± 10.6% and 69.0 ± 8.4%, respectively). This decreased embryonic development was due to the increased time required between the removal of the straws from the liquid nitrogen and the contact of the embryos with the warming medium when the warming was performed in a syringe in comparison with that for the warming in a dish. After verifying that the passage of VW embryos through the NET catheter does not have a damaging effect on their further in vitro development, the negative effect of warming in a syringe was also confirmed after NET. Fifteen fresh and SOPS-vitrified embryos warmed in a syringe or in a dish were transferred to each recipient (n = 28) and recovered 24 h later to assess their developmental progression. All embryos from the syringe group were found to have degenerated at recovery. The in vivo ES and HR from the dish group (80.4 ± 3.4% and 14.2 ± 7.2%, respectively) were lower (P < 0.05) than those from the fresh group (94.0 ± 4.1% and 36.8 ± 7.8%, respectively). Combining the warming in a dish and the NET procedure, 35 VW embryos were transferred to each of 10 gilts. Five recipients farrowed an average of 10.4 ± 0.9 piglets. In conclusion, the method of one-step warming in a syringe has a negative effect on the in vitro and in vivo viability of SOPS-vitrified porcine embryos. In addition, NET of SOPS-vitrified embryos warmed by the one-step method in a dish showed promising reproductive performance of recipients. However, despite the great potential of this technology, further developments are required for large-scale commercial applications.

Successful laparoscopic insemination with a very low number of flow cytometrically sorted boar sperm in field conditions.

The aim of this study was to develop a useful procedure for laparoscopic insemination (LI) with sex-sorted boar spermatozoa that yields adequate fertility results in farm conditions. In experiment 1, we evaluated the effects of single (oviducts) and double (oviducts and tips of the uterine horns) LI with X-sorted sperm on the reproductive performance of sows. Sows (N = 109) were inseminated once as follows: (1) single LI with 0.5 × 10(6) unsorted sperm per oviduct; (2) single LI with 0.5 × 10(6) sex-sorted sperm per oviduct; or (3) double LI with 0.5 × 10(6) sex-sorted sperm per oviduct and 0.5 × 10(6) sex-sorted sperm per uterine horn. The farrowing rates were lower (P < 0.05) in sows inseminated with sex-sorted sperm (43.2% and 61.9% for the single and double insemination groups, respectively) than in sows from the unsorted group (91.3%). Within the sex-sorted groups, the farrowing rate tended (P = 0.09) to be greater in sows inseminated using double LI. There were no differences in the litter size among groups. In experiment 2, we evaluated the effect of the number of sex-sorted sperm on the reproductive performance of sows when using double LI. Sows (N = 109) were inseminated with sex-sorted sperm once using double LI with: (1) 0.5 × 10(6) sperm per oviduct and 1 × 10(6) sperm per uterine horn; or (2) 1 × 10(6) sperm per oviduct and 2 × 10(6) sperm per uterine horn. Similarly high pregnancy (90%) and farrowing (80%) rates were achieved in both groups. The sows inseminated with the highest number of sperm tended (P = 0.09) to have more piglets (10.8 ± 0.7 vs. 9.2 ± 0.6). A high female proportion (number of female births divided by the total of all births ≥0.92) was obtained in both experiments using X-sorted sperm. Our results indicate that the double LI procedure, using between 3 and 6 × 10(6) sex-sorted sperm per sow produces adequate fertility at the farm level, making sperm-sexing technology potentially applicable in elite breeding units.

In vitro postwarming viability of vitrified porcine embryos: effect of cryostorage length.

Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN(2)) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN(2) has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage.

Nonsurgical deep uterine transfer of vitrified, in vivo -derived, porcine embryos is as effective as the default surgical approach

Surgical procedures are prevalent in porcine embryo transfer (ET) programs, where the use of vitrified embryos is quasi non-existent. This study compared the effectiveness of surgical vs nonsurgical deep uterine (NsDU) ET using vitrified, in vivo-derived embryos (morulae and blastocysts) on the reproductive performance and welfare of the recipients. The recipient sows (n = 122) were randomly assigned to one of the following groups: surgical ET with 30 vitrified-warmed embryos (S-30 group, control); NsDU-ET with 30 vitrified-warmed embryos (NsDU-30 group) and NsDU-ET with 40 vitrified-warmed embryos (NsDU-40 group). Regardless of embryo stage, the NsDU-ET with 40 embryos presented similar rates of farrowing (72.7%) and litter size (9.9 ± 2.1 piglets) as the customary surgical procedure (75.0% and 9.6 ± 2.7 piglets). Numbers of ET-embryos appeared relevant, since the NsDU-ET with 30 embryos resulted in a decrease (P < 0.05) in farrowing rates (38.9%) and litter sizes (5.7 ± 2.4 piglets). In conclusion, we demonstrate for the first time that farrowing rate and litter size following a NsDU-ET procedure increase in function of a larger number of transferred vitrified embryos, with fertility equalizing that obtained with the invasive surgical approach. The results open new possibilities for the widespread use of non-invasive ET in pigs.

Effects of Hoechst 33342 staining and ultraviolet irradiation on the developmental competence of in vitro-matured porcine oocytes.

Hoechst 33342 (H342) in combination with ultraviolet (UV) irradiation is frequently used to assist the enucleation of porcine oocytes in somatic cell nuclear transfer programs. This work evaluated the effects of H342 (5 μg/mL for 12 min) staining and/or exposure to UV irradiation on fertilisability and developmental capacity of porcine oocytes matured in vitro. In Experiment 1, a total of 1388 mature oocytes were distributed in the following groups: Group 1: oocytes without treatment (Control), Group 2: oocytes stained with H342, Group 3: oocytes stained with H342 and UV irradiated for 30 sec, and Group 4: oocytes UV irradiated for 30 sec. Oocytes from each group were exposed to thawed spermatozoa and cultured for 18 h to assess fertilization parameters or for 7 d to evaluate embryo development. Sperm penetration (P < 0.001) and monospermy (P < 0.04) were lower in oocytes exposed to H342/UV (80.7 ± 4.5% and 30.7 ± 5.4%, respectively) than in oocytes from the control group (94.9 ± 4.3 and 50.0 ± 4.9, respectively). The oocytes exposed to H342/UV showed lower (P < 0.001) cleavage (49.8 ± 2.9%) and blastocyst (7.7 ± 2.9%) rates than oocytes from the other groups (range: 73.8 ± 2.9% to 77.7 ± 2.9% and 22.3 ± 2.9% to 30.9 ± 3.0%, respectively). Experiment 2 was designed to evaluate the effect of shorter UV irradiation (5 sec). A total of 1835 mature oocytes were separated into the same groups as those of Experiment 1. The fertilization parameters and the cleavage rates were not influenced by the different treatments. However, the oocytes exposed to H342 and UV irradiation for 5 sec showed a lower (P < 0.02) rate of blastocyst formation (15.2 ± 4.5%) than the oocytes from other groups (range: 26.1 ± 4.5% to 30.7 ± 4.5%). In conclusion, our results demonstrate that the combination of H342 staining with UV irradiation has a clear deleterious effect on the developmental ability of oocytes, with the effects being more intense with increased exposure to UV irradiation.