M.A. Angel

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.

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.

Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability

The objective of this study was to develop an adequate sperm handling protocol in order to obtain a sex-sorted sperm population with an optimal fertilizing ability. For this purpose, different aspects of the sorting procedure were examined. The effects of the high dilution rates (experiment 1), type of collection medium used (experiment 2), and sheath fluid composition (experiment 3) on sorted boar sperm quality and function were evaluated. Sperm quality was assessed by motility and viability tests, whereas sperm function was evaluated by an in vitro fertilization assay which determined the penetration and polyspermy rates as well as the mean number of sperm penetrating each oocyte. In experiment 1, the results obtained indicated that the high dilution rates did not cause a decrease either in the sperm quality parameters evaluated or the in vitro fertilization ability of spermatozoa. In experiment 2, although sperm quality was not affected, fertilizing ability was compromised after sorting, regardless of the collection medium that was used. In the experiment 3, all groups displayed adequate sperm quality values, but higher in vitro fertility parameters were obtained for spermatozoa sorted in presence of EDTA in the sheath fluid and egg yolk (EY) in the collection media when compared with those sorted in absence of these protective agents. No differences in penetration rates between unsorted highly diluted (control) and sorted sperm in the presence of EDTA and EY were observed. In conclusion, fertilizing ability was compromised in sex-sorted sperm. The addition of EDTA to sheath fluid and EY to collection medium improved boar sperm fertilizing ability, and both agents should be included as essential media components in future studies.

The in vitro and in vivo developmental capacity of selected porcine monospermic zygotes

In this study, we evaluated the in vitro and in vivo developmental capacity of selected monospermic zygotes produced in vitro. Cumulus-oocyte complexes were matured in vitro and inseminated with frozen-thawed spermatozoa. Thirteen hours after insemination, presumptive zygotes were centrifuged at 15,000 ×g for 20 minutes to polarize the lipids in the cytoplasm and permit the visualization of pronuclei. Then, the oocytes were individually classified as bipronuclear (2PN) or polypronuclear (three or more pronuclei, PPN). To examine embryo development, 102 selected zygotes were cultured for 7 days. There were no differences in cleavage rate (93.0% and 88.9% for 2PN and PPN zygotes, respectively). However, the blastocyst formation rate was higher (P < 0.003) in 2PN (80.7%) zygotes than in PPN (53.3%) zygotes. The control (noncentrifuged, nonselected zygotes) group showed lower (P < 0.003) cleavage rate and blastocyst formation than the 2PN and PPN zygotes. In a second experiment, 2PN zygotes and control zygotes were transferred (30 zygotes per transfer) by laparoscopy into the oviducts of recipient gilts (10 recipients per group) on the first day of standing estrus. The farrowing rates were 70% and 40% for transfers made with 2PN and control zygotes, respectively. The average number of piglets born per recipient farrowed did not differ between groups (4.9 ± 0.6 and 4.5 ± 1.2, respectively), but the efficiency (number of live piglets per total transferred embryos) was higher (P < 0.01) for 2PN zygotes than for the control group (9.3% and 4.0%, respectively). These results demonstrate the effectiveness of centrifugation for the selection of monospermic zygotes as a procedure to improve in vitro embryo production in pigs. In addition, the results indicate that the laparoscopic technique described here is a simple and effective procedure for transferring embryos into one oviduct.

An earlier uterine environment favors the in vivo development of fresh pig morulae and blastocysts transferred by a nonsurgical deep-uterine method.

This study aimed to evaluate the effect of recipient-donor estrous cycle synchrony on recipient reproductive performance after nonsurgical deep-uterine (NsDU) embryo transfer (ET). The transfers (N=132) were conducted in recipients sows that started estrus 24 h before (–24 h; N=9) or 0 h (synchronous; N=31), 24 h (+24 h; N=74) or 48 h (+48 h; N=18) after the donors. A total of 30 day 5 morulae or day 6 blastocysts (day 0=onset of estrus) were transferred per recipient. The highest farrowing rates (FRs) were achieved when estrus appeared in recipients 24 h later than that in the donors (81.1%), regardless of the embryonic stage used for the transfers. The FR notably decreased (P<0.05) when recipients were –24 h asynchronous (0%), synchronous (61.3%) or +48 h asynchronous (50%) relative to the donors. No differences in litter size (LS) and piglet birth weights were observed among the synchronous and +24 h or +48 h asynchronous groups. While a +24 h asynchronous recipient was suitable for transfers performed with either morulae (FR, 74.3%; LS, 9.2 ± 0.6 piglets) or blastocysts (FR, 84.6%; LS, 9.8 ± 0.6 piglets), a + 48 h asynchronous recipient was adequate for blastocysts (FR, 87.5%; LS, 10.4 ± 0.7 piglets) but not for morulae (FR, 30.0%; LS, 7.3 ± 2.3 piglets). In conclusion, this study confirms the effectiveness of the NsDU-ET technology and shows that porcine embryos tolerate better a less advanced uterine environment if they are nonsurgically transferred deep into the uterine horn.

Forskolin improves the cryosurvival of in vivo-derived porcine embryos at very early stages using two vitrification methods

This study was aimed to determine the effect of forskolin on the viability of in vivo-derived porcine embryos vitrified by the superfine open pulled straw (SOPS) or solid surface vitrification (SSV) methods at the 2-cell, 4-cell, and blastocyst stages. Zygotes, 2- to 4-cell embryos, and morulae were obtained from superovulated sows. After collection, embryos were cultured for 24h with 0 or 10 μM forskolin and then vitrified using the SOPS and SSV method, or not vitrified (fresh controls). Fresh and vitrified-warmed 2-cells, 4-cells, and blastocysts were cultured for additional 96 h, 72 h and 24 h, respectively. At the end of the culture, embryos were evaluated for progression to the blastocyst stage and total cell number. The vitrification method did not affect any of the parameters evaluated for any embryo stage. Forskolin increased (P<0.01) the blastocyst formation and the final developmental stage of vitrified 2- and 4-cell embryos. However, these embryos exhibited lower (P<0.003) blastocyst formation rates than their fresh counterparts. The total cell number and hatching rate were similar in both groups (vitrified and fresh) of 2- and 4-cell embryos. Vitrified blastocysts exhibited viabilities, final developmental stages, hatching rates, and total cell numbers that were similar to those of their fresh counterparts, regardless of the addition of forskolin. In conclusion, the SOPS and SSV methods are suitable for the cryopreservation of in vivo-derived 2- to 4-cell porcine embryos. Pre-treatment with forskolin for 24h before vitrification improves the cryotolerance of 2- and 4-cell porcine embryos.

Effects of lipid polarisation on survival of in vivo-derived porcine zygotes vitrified by the superfine open pulled-straw method

This study aimed to evaluate the post-warming in vitro viability of intact porcine zygotes vitrified using the superfine open pulled-straw (SOPS) method and to investigate whether cryotolerance is increased by lipid polarisation before vitrification. In vivo-derived zygotes (n=317) were either untreated before SOPS vitrification or subjected to one of the following pre-treatments: (1) centrifugation (20 min, 15000 g) or (2) equilibration in high-osmolality medium (6 min, 400 mOsm kg(-1)) followed by centrifugation. Vitrified-warmed and non-vitrified fresh zygotes were cultured in vitro for 120 h. There were no differences in the blastocyst formation rates between the vitrification groups (from 35.4±5.3% to 48.2±5.6%), but fresh zygotes exhibited higher (P<0.001) blastocyst formation rates (87.5±5.3%) than did vitrified-warmed zygotes. The total blastocyst cell number was similar among all groups (from 34.9±2.8 to 44.1±2.8). In conclusion, SOPS vitrification is a promising method for the cryopreservation of untreated in vivo-derived porcine zygotes. Neither lipid polarisation by centrifugation nor exposure to a high-osmolality medium followed by centrifugation affected the post-warming in vitro viability of zygotes. Our study also demonstrated that the donor is an important factor in determining the success of vitrification for in vivo-derived porcine zygotes.