Achievements and future perspectives of embryo transfer technology in pigs.

Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non-surgical ET and short- and long-term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non-surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short-term use in pig production.

The battle of the sexes starts in the oviduct: modulation of oviductal transcriptome by X and Y-bearing spermatozoa.

BACKGROUND: Sex allocation of offspring in mammals is usually considered as a matter of chance, being dependent on whether an X- or a Y-chromosome-bearing spermatozoon reaches the oocyte first. Here we investigated the alternative possibility, namely that the oviducts can recognise X- and Y- spermatozoa, and may thus be able to bias the offspring sex ratio. RESULTS: By introducing X- or Y-sperm populations into the two separate oviducts of single female pigs using bilateral laparoscopic insemination we found that the spermatozoa did indeed elicit sex-specific transcriptomic responses. Microarray analysis revealed that 501 were consistently altered (P-value < 0.05) in the oviduct in the presence of Y-chromosome-bearing spermatozoa compared to the presence of X-chromosome-bearing spermatozoa. From these 501 transcripts, 271 transcripts (54.1%) were down-regulated and 230 transcripts (45.9%) were up-regulated when the Y- chromosome-bearing spermatozoa was present in the oviduct. Our data showed that local immune responses specific to each sperm type were elicited within the oviduct. In addition, either type of spermatozoa elicits sex-specific signal transduction signalling by oviductal cells. CONCLUSIONS: Our data suggest that the oviduct functions as a biological sensor that screens the spermatozoon, and then responds by modifying the oviductal environment. We hypothesize that there might exist a gender biasing mechanism controlled by the female.

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.

Effects of Hoechst 33342 staining and ultraviolet irradiation on mitochondrial distribution and DNA copy number in porcine oocytes and preimplantation embryos.

Hoechst 33342 (H342), in combination with ultraviolet (UV) irradiation, is frequently used to aid or confirm the enucleation of porcine oocytes in somatic cell nuclear transfer programs. The exposure of oocytes to H342 and UV irradiation has a deleterious effect on the development of in vitro-fertilized porcine oocytes, with increasing exposure to UV irradiation (up to 30 sec) having more drastic effects. It has been hypothesized that this decrease in embryonic development could be due to damage to the mitochondrial DNA (mtDNA). To investigate this hypothesis, we analyzed the mitochondrial distribution and DNA copy number of in vitro-matured porcine oocytes exposed to H342/UV and the subsequent embryonic development compared with the mitochondrial distribution and DNA copy number of in vivo-derived oocytes and embryos. Using quantitative, real-time polymerase chain reaction (qPCR) protocols to analyze mtDNA and confocal laser scanning microscopy with MitoTracker Deep Red to determine mitochondrial distribution, we demonstrated that the simultaneous exposure of in vitro-matured porcine oocytes to H342 staining and UV irradiation is associated with reduced oocyte developmental competence and abnormal mitochondrial distribution in the resulting cleaved embryos. In addition, 2- to 4-cell embryos derived from oocytes exposed to H342/UV showed a significant decrease in mtDNA copy number. These results should be considered when H342/UV procedure is used during nuclear transfer in recipient porcine oocytes.

Characterization of glycoside residues of porcine zona pellucida and ooplasm during follicular development and atresia.

The glycoside residues (glycoconjugates, GC) of the zona pellucida (ZP) glycoproteins are important during the first phases of fecundation. Our aim in this work was to determine the lectin affinity pattern of porcine ZP in order to analyze the changes that take place during: (a) preantral folliculogenesis, (b) the follicular atresia process, and (c) antral growth. Several prepubertal and adult pig ovaries and different sized antral follicles were used. Conventional carbohydrate histochemical techniques and peroxidase and digoxigenin (DIG) lectins were used to reveal the acid groups and the glycosidic residues of the ZP. It was seen that the ZP forms in the preantral follicles throughout their growth period. In primordial and primary follicles, ZP in the process of formation showed neutral GC. SBA, RCA-I, MAA, WGA lectins, and AAA after methylation-saponification (MS) were positive in the ZP of primordial and primary follicles. The affinity for SBA, RCA-I, MAA, and WGA increased in the multilaminar-primary follicles and new affinities for UEA-I and LFA were observed. After MS, AAA, SNA, PNA, and SBA reactivity was observed. The ZP of antral follicle oocytes of different sizes showed the same lectin pattern as multilaminar-primary follicles. The oocyte ooplasm and the follicular fluid of large antral follicles showed less affinity for WGA and LFA lectins and less intensive staining with AB (pH 2.5). Atresia did not change the antral or preantral follicle oocyte ZP lectin pattern. In conclusion, the follicles showed substantial changes in their ZP glycosidic composition as they developed, especially, during the change from primary to multilaminar-primary follicles. The ZP glycosidic composition showed no significant change during the growth of antral follicles and follicular atresia in our study.

Influence of insemination time on the fertility of sex sorted frozen-thawed Y-sperm in red deer.

The aim of this study was to assess the effect of insemination timing on pregnancy rates in red deer (Cervus elaphus) when using sex-sorted sperm samples. Semen was collected by electroejaculation from 8 mature stags and processed to obtain: Conventional samples, following standard freezing procedures for commercial purposes; Control sorted samples, diluted and handled as per sorted samples but without being submitted to the sorter passage; and Y Sex Sorted (YSS) samples. Hinds were synchronized via intravaginal CIDR (Controlled Internal Drug Release) placement and given eCG (Folligon® PMSG Serum Gonadotrophin) on day 12, upon CIDR removal. They were then inseminated with one of each sperm treatment, at the following post-eCG intervals: I_1, 55:01-55:30 h; I_2, 55:31-56:00 h; I_3, 56:01-56:30 h; or, I_4, 56:31-57:00 h. Pregnancy rates were assessed at parturition. Average pregnancy rates were highest (P < 0.05) for Conventional samples (77.6%), but similar between YSS (49.8%) and Control sorted (51.3%) samples. However, when insemination interval was taken into account, pregnancy rates within the YSS group, pregnancy rates were 80 and 83.1% for I_1 and I_2, respectively were obtained. Notably, these rates were similar (P > 0.05) to the average pregnancy rates obtained with Conventional samples (77.6%). As expected, YSS sperm yielded 94% male offspring contrasting with the 57% males obtained with Conventional and Control sorted samples. Our findings support the importance of developing specific insemination timing protocols to improve pregnancy rates when using frozen-thawed sex-sorted sperm. These findings provide the foundation for further investigations in order to determine why the YSS sperm are able to fertilize the oocyte in a shorter period of time than the conventional samples.

Optimization of protocols for Iberian red deer (Cervus elaphus hispanicus) sperm handling before sex sorting by flow cytometry.

Currently, sperm reproductive biotechnologies such as sex sorting and cryopreservation are undoubtedly valuable tools for improving the economic and biological efficiency of red deer production systems. In this context, and because of the particular characteristics of this species (extensive exploitation typically far from laboratory facilities), a key goal is to optimize the design of an adequate handling protocol of sperm samples before samples are subjected to sex sorting and cryopreservation procedures to obtain better outputs from the application of these technologies. The main aim of this paper was to design an adequate protocol for Iberian red deer sperm handling before sex sorting by flow cytometry to obtain optimal yields when sex sorting is used in this species. Semen samples from 11 adult males were obtained by electroejaculation during the breeding season. In this study, we tested different protocols for the handling of Iberian red deer spermatozoa before sorting by using different concentrations of sperm (400 or 800 × 106) and adding or not Hoechst 33342 before the transport of samples to the sorting facilities. Based on the results, the most adequate method used to handle samples before sorting was transportation at a high sperm concentration (800 × 106/mL) without Hoechst 33342. These transportation conditions in combination with Hoechst 33342 staining at 5.2 µL/mL once at the flow cytometry laboratory resulted in better (P < 0.05) sorting efficiency (99.9% of the samples showing split) than both, those samples transported at 400 × 106sperm/mL (between 51.2 and 55.2% of the samples showing split) and those samples stained before transport at a sperm concentration of 400 × 106sperm/mL (between 15.4 and 75.7% of the samples showing split). Sorting rates and sperm quality after sorting and cryopreservation was not affected (P > 0.05) by sperm handling before sorting. Moreover, the sorting yields were compatible with the practical application of these reproductive biotechnologies.

Improving the efficiency of sperm technologies in pigs: the value of deep intrauterine insemination.

The use of AI in pigs has dramatically expanded in the last few years. New methodological advances in AI are required to serve the requirements of new sperm technologies, such as the use of low dose AI, because the use of cervical AI has a very low efficiency leading to low fertility results. One of the strategies devised to meet these requirements is the deposition of semen near the site of fertilization in the oviduct. Using deep intrauterine insemination with a specially designed catheter, a 20-fold reduction in the number of freshly and diluted inseminated spermatozoa can be achieved without decreasing farrowing rates. Moreover, an advantage of deep intrauterine insemination is the possibility of using processed, 'weaker' spermatozoa such as those that have been frozen-thawed or sex-sorted. Although deep intrauterine insemination should be of benefit to the pig industry, more investigations are needed to understand the mechanisms related to sperm colonization of the oviducts and identify the minimal sperm numbers needed to obtain maximal fertility results for processed and unprocessed boar spermatozoa.

Effects of centrifugation before freezing on boar sperm cryosurvival.

Current protocols for boar sperm cryopreservation require the centrifugation of semen in order to separate sperm cells from the seminal plasma. This study evaluated the influence of different centrifugation regimes on both sperm recovery and yield (percentage of viable sperm with an intact acrosome relative to the initial sperm population) after centrifugation (experiment 1) as well as the influence of different centrifugation regimes on boar sperm cryosurvival (experiment 2). In both experiments, sperm-rich fractions from 3 boars were diluted, pooled, and cooled to 17 degrees C before centrifugation. In experiment 1, the g-forces tested were 400, 800, 1600, and 2400 x g for 3 or 5 minutes, using the standard regime (800 x g for 10 minutes) as a reference. Sperm recovery (Bürker Chamber) and yield (triple fluorescent stain of PI/R123/FITC-PNA [DNA-specific fluorochrome propidium iodide/mitochondria-specific fluorochrome rhodamine-123/acrosome-specific fluorochrome fluorescein isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin]) were calculated. The highest recovery and yield (P <.05) values were achieved using 2400 x g for 5 or 3 minutes and 1600 x g for 5 minutes, which showed no differences (P >.05) from the reference in terms of sperm yield. In experiment 2, cooled semen was centrifuged using 3 different regimes: C1 (2400 x g for 3 minutes), C2 (1600 x g for 5 minutes), and C3 (800 x g for 10 minutes). Pellets were diluted in lactose-egg yolk (LEY)-glycerol-Equex STM (1 x 10(9) cells/mL) and frozen in 0.5-mL straws. After thawing, sperm quality was assessed after 30 and 150 minutes of incubation (37 degrees C). Centrifugation regimes C1 and C2 showed significantly (P <.05) higher postthaw sperm motility (assessed with a computer-assisted semen analysis system), viability (evaluated as for experiment 1), and percentage of uncapacitated sperm (assessed with a chlortetracycline assay) than did C3. In addition, C1 had the highest (P <.05) oocyte penetrating ability (assessed with the homologous in vitro penetration test performed with immature oocytes). Malondialdehyde production, assessed with the thiobarbituric acid reactive species test, was unaffected (P >.05) by the centrifugation regime used. We conclude that high g-force (2400 x g) and short centrifugation time (3 minutes) do not affect sperm recovery and yield and that, moreover, they have a positive effect on the cryosurvival of boar sperm. Therefore, we recommend the use of short-term centrifugation with a relatively high g-force (2400 x g for 3 minutes) in boar sperm cryopreservation protocol.

Birth of piglets after deep intrauterine insemination with flow cytometrically sorted boar spermatozoa.

The present study was carried out to determine the pregnancy rates, farrowing rates and litter size in sows with either induced or spontaneous ovulation inseminated with flow cytometric sorted spermatozoa using deep intrauterine insemination technology. Spermatozoa were stained with Hoechst 33342 and sorted by flow cytometry/cell sorting but not separated into separate X and Y populations. In Experiment 1, sows (n=200) were weaned and treated for estrus/ovulation induction with eCG/hCG. Inseminations with either sorted (70 or 140 million) or non-sorted (70 or 140 million) spermatozoa were done using a specially designed flexible catheter. Farrowing rates were 39.1 and 78.7% for 70 million of sorted and non-sorted, respectively, and 46.6 and 85.7% for 140 million of sorted and non-sorted, respectively (P<0.05). The litter size in sows inseminated with sorted spermatozoa showed a tendency to be lower than when non-sorted spermatozoa were inseminated. In Experiment 2, sows (n=140) were inseminated as in Experiment 1 except that natural estrus was used. The ovaries of these sows were evaluated by transrectal ultrasonography. Farrowing rates were 25 and 77.2% for 70 million of sorted and non-sorted, respectively, and 32 and 80.9% for 140 million of sorted and non-sorted, respectively (P<0.05). These results show that the Deep Intrauterine Insemination technology can be successfully used to produce piglets from sorted spermatozoa when sows are hormonally treated to induce synchronous post weaning oestrus and ovulation.

Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa.

The present study evaluates the effectiveness of the transcervical deep intrauterine insemination (DUI) with a reduced number of frozen-thawed boar spermatozoa in weaned sows. DUI was performed using a specially designed flexible device (length 180 cm, outer diameter 4mm, working channel 1.8mm, working channel's volume 1.5 ml) that was inserted through an artificial insemination spirette to cross the cervix lumen and moved into one uterine horn as far as possible. Spermatozoa diluted in 7.5 ml of BTS were flushed into the uterine horn by a syringe attached to the working channel. In Experiment 1, 111 hormonally treated (eCG/hCG) weaned sows were inseminated once using one of the following three regimens: (1) DUI with frozen-thawed spermatozoa (1000 x 10(6) cells per dose; n=49); (2) DUI with fresh semen (150 x 10(6) cells per dose; n=29, as control of DUI procedure); and (3) cervical insemination with frozen-thawed spermatozoa (6000 x 10(6) cells diluted in 100ml; n=33). No differences (P>0.05) were found for farrowing rates (77.55, 82.76, and 75.76, respectively) or litter sizes (9.31+/-0.41, 9.96+/-0.32, and 9.60+/-0.53 piglets born per litter, respectively) among the groups. In Experiment 2, DUI was performed on the spontaneous estrus in weaned sows (2-6 parity) with 1000 x 10(6) frozen-thawed (40 sows) or 150 x 10(6) fresh spermatozoa (38 sows). The farrowing rate of sows inseminated twice with frozen-thawed spermatozoa (70%) was significantly (P<0.05) lower than with fresh semen (84.21%). No significant difference (P>0.05) was found in litter size between frozen-thawed spermatozoa (9.25+/-0.23 piglets born per litter) and fresh semen (9.88+/-0.21 piglets born per litter). These preliminary results indicate that application of DUI provides acceptable fertility in weaned sows using a relatively low number of frozen-thawed spermatozoa.

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.

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.

Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars.

A major limiting factor for commercial cryopreservation of boar semen for artificial insemination (AI) is the large individual variation to cooling, where the degree of cell dehydration during ice (re)shaping seems to play a major role. This study investigated, in the frozen state, the degree of dehydration and ice crystal distribution in boar semen doses whose spermatozoa displayed different viability after thawing. Cross-sectioned medium-straws (0.5 mL, n=10) from a total of 10 stud boars classified as "good"(n=5) or sub-standard (e.g., "bad" freezers, n=5) by conventional analyses (computer assisted motility and sperm viability) were examined by Cryo-scanning electron microscopy (Cryo-SEM) to determine whether differences between groups could be already distinguishable prior to thawing. The degree of hydration was monitored in relation to the areas of ice crystal formed extracellularly (lakes), the areas of frozen, concentrated extender (veins) where spermatozoa were located and the degree of compartmentalization (number of lakes) present. Irrespectively of the region studied, the gradient of main dehydration (as lakes) observed along the cross-section area of the straws was very irregular. Most spermatozoa were enclosed in the freezing extender matrix and no obvious signs of external membrane damage were observed. None of the Cryo-SEM variables significantly correlated with post-thaw sperm parameters (p>0.05). However, we identified significant differences (p<0.0001) among boars for all ultrastructure variables studied, including the size of the veins, where differences in solute concentration is expected. We concluded that despite the large variability in ice crystal formation during the conventional freezing process among boars, this is unrelated to inter-boar post-thaw sperm differences.