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SUMMARY It is well documented that oocytes from small antral follicles are less competent than those derived from large follicles, and we have previously shown that glial cell line-derived neurotrophic factor (GDNF) enhances developmental competence in oocytes from antral follicles. Exactly how GDNF effects this change and if it depends on the stage of oocyte development is currently unknown. The objective of this study was to examine the transcriptomic effects of follicle size and GDNF on the in vitro maturation of porcine oocytes. Microarray analysis uncovered differentially expressed transcripts among in vitro-matured porcine oocytes from different-size antral follicles, in the absence or presence of GDNF. Oocytes isolated from small follicles showed a lower state of maturation than those from large follicles, with several transcripts associated with meiotic arrest. Addition of GDNF to the culture media had effects that depended on the stage of the follicle from which the oocyte was isolated, with those from small follicles showing decreased expression of genes associated with acetyltransferase activity while those from large follicles showed decreased metabolic activity. In summary, our results revealed considerable differences between the transcriptomes of small- and large-follicle-derived oocytes. Furthermore, GDNF affects the developmental competence of oocytes in follicle-stage dependent manner. Thus, improving our understanding of the requirements for successful in vitro maturation of porcine oocytes will inform current reproductive technologies, with implications for the future of animal and human health. Mol. Reprod. Dev. 2014. © 2014 Wiley Periodicals, Inc.

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

This study was conducted to compare the efficacy of four in vitro fertilization (IVF) media: Bracket and Oliphant's medium (BO), modified medium 199 (IVF-M199), modified Tyrode's medium (MTM), and modified KSOM (m-KSOM) on fertilization efficiency and blastocyst formation rate. In addition, we wanted to investigate the benefit of prolonging the IVF period (from 6 to 18 h) using the two most effective IVF media determined in our initial experiment; subsequently, blastocyst viability was assessed following vitrification. A higher incidence of polyspermic fertilization was observed in the MTM (6%) and in BO, in both the 6 and 18 h (7% and 11%, respectively) groups, than in the m-KSOM (1%) or in the IVF-M199 6 or 18 h (1 and 3%, respectively) groups. Cleavage rates were similar in BO, IVF-M199, and MTM 48 h post-fertilization; however, the lowest cleavage rate was observed for m-KSOM. A greater proportion of zygotes developed into 8-cell embryos in IVF-M199 than in other IVF media. Subsequently, a greater proportion of blastocyst formation and hatching was achieved in IVF-M199 (40% and 79%, respectively) or BO (35% and 74%, respectively) than in m-KSOM (18% and 58%, respectively) or MTM (22% and 66%, respectively). Prolonging IVF to 18 h did not alter cleavage rates; however, the highest rate of overall blastocyst formation was achieved in the IVF-M199 18 h (49%), rather than in the BO 18 h (20%) group. Vitrified/thawed blastocysts from IVF-M199 groups re-expanded and developed better, as compared to the BO 18 h group, and hatching rate and total cell number in IVF-M199 18 h group was comparable to the control groups (non-vitrified). Vitrification reduced survival compared to controls. In conclusion, IVF-M199 was successfully used for IVF, compared favorably to BO medium, and offered the advantage of an extended IVF period for up to 18 h that requires only one-half a dose of semen, and resulted in better quality blastocysts that endured vitrification with a hatching rate comparable to that of control groups.

The purpose of this study was to evaluate the effect of cooling ovarian tissue on pig pre-antral follicles. Ovaries were maintained in saline solution (0.9%) at 4 or 20 degrees C for 6, 12 or 18 h. After storage, pre-antral follicles were morphologically evaluated. While primordial follicles were not affected by the storage, the percentage of morphologically normal growing follicles was significantly reduced in ovarian tissue stored at 20 degrees C for 12 or 18 h. To test the viability of stored follicles, growing follicles isolated from ovaries stored at 4 degrees C for 18 h and at 20 degrees C for 6 h were cultured for 3 days. Follicles stored in either condition presented the same growth pattern in vitro as fresh follicles. We conclude that storage of pig ovaries at 4 degrees C for up to 18 h or at 20 degrees C for up to 6 h does not affect the morphology of growing follicles or their ability to grow in vitro.

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g., gene knock-in, knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs, microinjection of piPSCs into embryos, embryo transfer and production of chimeric animals based on successful protocols.

Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.

Factors influencing pig oocyte activation by electrical stimulation were evaluated by their effect on the development of parthenogenetic embryos to the blastocyst stage to establish an effective activation protocol for pig nuclear transfer. This evaluation included 1) a comparison of the effect of epidermal growth factor and amino acids in maturation medium, 2) an investigation of interactions among oocyte age, applied voltage field strength, electrical pulse number, and pulse duration, and 3) a karyotype analysis of the parthenogenetic blastocysts yielded by an optimized protocol based on an in vitro system of oocyte maturation and embryo culture. In the first study, addition of amino acids in maturation medium was beneficial for the developmental competence of activated oocytes. In the second study, the developmental response of activated oocytes was dependent on interactions between oocyte age at activation and applied voltage field strength, voltage field strength and pulse number, and pulse number and duration. The formation of parthenogenetic blastocysts was optimal when activation was at 44 h of maturation using three 80-μsec consecutive pulses of 1.0 kV/cm DC. Approximately 84% of parthenogenetic blastocysts yielded by this protocol were diploid, implying a potential for further in vivo development.

This study was conducted to determine fertilization rate and embryo development using the Beltsville Sperm Sexing Technology with two different laser power outputs, 25 and 125 milliwatts (mW). Freshly ejaculated boar semen was diluted; one aliquot was not stained or sorted (nonsort) and a second aliquot was stained with Hoechst 33342 and sorted as a complete population, not separated into X and Y populations (all-sort). Ovulation controlled gilts were surgically inseminated with 2 x 10(5) spermatozoa (44-46 hr after human chorionic gonadotropin (hCG)) into the isthmus of each oviduct, one oviduct receiving nonsort and the other all-sort at 25 or 125 mW. A total of 426 embryos were flushed from oviducts at slaughter 43 hr after laparotomy and prepared for determination of fertilization and cleavage rates using confocal laser microscopy for analysis of actin cytoskeleton and chromatin configuration. The percentage of fertilized eggs and embryos was less for the 25 mW all-sort compared to nonsort or the 125 mW all-sort (77.9 vs. 96.3 and 96.2%, P0.05). The percentage of fragmented embryos was greater for the 25 mW all-sort than the nonsort (15.2 vs. 4.5%, P0.05), but did not differ significantly from 125 mW all-sort mean (7.2%). The percentage of normal embryos (80.4% overall) did not differ (P0.05) among treatments. However, the rate of embryo development was slower (P0.05) after insemination with the 25 mW all-sort spermatozoa compared to nonsort spermatozoa. Embryos in the 3-4 and 5-9 cell stages for the 25-mW all-sort and nonsort were 78 and 20% vs. 49 and 50%, respectively. The embryo percentages for the 125 mW (3-4 and 5-9 cell stages, 59 and 35%) did not differ significantly (P0.05) from the nonsort or 25 mW all-sort. We conclude that the use of 125 mW laser power for sorting boar spermatozoa is advantageous to maintain high resolution separation and has no detrimental effect on embryo development compared to 25 mW.

Pig embryos suffer severe sensitivity to hypothermic conditions, which limits their ability to withstand conventional cryopreservation. Research has focused on high lipid content of pig embryos and its role in hypothermic sensitivity, while little research has been conducted on structural damage. Documenting cytoskeletal disruption provides information on embryonic sensitivity and cellular response to cryopreservation. The objectives of this study were to document microfilament (MF) alterations during swine embryo vitrification, to utilize an MF inhibitor during cryopreservation to stabilize MF, and to determine the developmental competence of cytoskeletal-stabilized and vitrified pig embryos. Vitrified morulae/early blastocysts displayed MF disruptions and lacked developmental competence after cryopreservation; hatched blastocysts displayed variable MF disruption and developmental competence. Cytochalasin-b did not improve morula/early blastocyst viability after vitrification; however, it significantly (P < 0.05) improved survival and development of expanded and hatched blastocysts. After embryo transfer, we achieved pregnancy rates of almost 60%, and litter sizes improved from 5 to 7.25 piglets per litter. This study shows that the pig embryo cytoskeleton can be affected by vitrification and that MF depolymerization prior to vitrification improves blastocyst developmental competence after cryopreservation. After transfer, vitrified embryos can produce live, healthy piglets that grow normally and when mature are of excellent fecundity.

In vitro-produced embryos exhibit decreased cell numbers, small inner cell masses and reduced pregnancy rates after transfer. Evaluation of intracellular components of in vitro-produced or -manipu- lated embryos will lead to improved methodology for embryo production. Whole mount techniques were developed to utilize terminal deoxynucleotidyl-transfer- ase 38 nick end labeling (TUNEL) to detect broken DNA. Subsequent labeling of either tubulin or actin filaments provides further evidence of cytological damage. Por- cine embryos produced in vitro or in vivo were evalu- ated throughout the cleavage and preimplantation stages of development. Early cleavage stages up to the 8-cell stage never contained TUNEL-labeled nuclei. However, TUNEL labeling of in vitro-produced morula revealed some blastomeres with broken DNA. Nearly all in vitro-produced blastocysts displayed some TUNEL positive cells, whereas in vivo-collected embryos at a similar stage displayed few, if any, TUNEL-labeled nuclei. The ratio of TUNEL-labeled DNA to total DNA area of in vitro-derived blastocysts was significantly greater than their in vivo counterparts (P , 0.05). Microtubule and microfilament labeling identified blasto- meres of unequal size and shape that were losing cellular integrity. These data suggest that the combina- tion of these labeling techniques may be useful in evaluating cellular damage in embryos produced under in vitro conditions. Mol. Reprod. Dev. 51:59-65, 1998. r 1998 Wiley-Liss, Inc.

The in vitro production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from in vitro Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than in vivo–derived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of in vivo (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The “eIF2 signalling”, “mitochondrial dysfunction”, “regulation of eIF4 and p70S6K signalling”, “protein ubiquitination”, and “mTOR signalling” pathways were down-regulated in PA HB. Dysregulation of notch signalling–associated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (NANOG, GATA2, KRT8, LGMN, and DPP4) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.

Media are available that can deliver modest porcine embryonic development from a single-cell zygote to the blastocyst stage. However, few embryos develop to hatched blastocysts by Day 7 in vitro, indicating deficiencies in media that inhibit early embryonic development. A defined culture medium, Beltsville Embryo Culture Medium (BECM-3), was developed to support porcine zygote development to the blastocyst stage. When fetal bovine serum was added by late Day 5 (late morula/early blastocyst stage), 80% of total embryos cultured from Day 2 developed into hatched blastocysts by Day 8. There was also a significant increase in the mean cell number of blastocysts and hatched blastocysts when culture was performed in BECM-3-based media in the absence of BSA fraction V. These studies provide a chemically defined foundation for elucidating key developmental components of preimplantation pig embryos.

Considerable progress has been made in the improvement and simplification of cryopreservation procedures routinely used in embryo transfer programs. Conventional slow-rate, programmable freezing and vitrification of embryos have given veterinarians, scientists and producers alternatives in their herd reproduction practices, however, pregnancy rates after cryopreservation are not equivalent to fresh embryo transfer. No matter how embryos are cryopreserved, some always die, and that can be costly to the producer. Documenting cellular damage during or after cryopreservation would provide useful, non-empirical information for understanding cellular sensitivities to cryopreservation and will lead to improved protocols for embryo cryopreservation and a better understanding of domestic animal embryology. This paper discusses the current progress in embryo cryopreservation, and will address intracellular damage to the cytoarchitecture of cryopreserved embryos and attempts to detour this damage by utilizing cytoskeletal stabilization prior to cryopreservation.

Summary Assessing the functional capacity of sperm in vitro is of particular interest to the artificial insemination industry in order to select sires with the best fertilizing capacity. Early efforts to evaluate semen dating into the past century were based on microscopical observation of motility and abnormal morphology. Frequently, eosin stain was used to quantify live and dead sperm while giemsa stain was used for morphological evaluation. These methods along with visual inspection of the semen were useful but their repeatability was questionable. The advent of fluorescent staining improved microscopical evaluation of sperm, however, one of the major drawbacks remains, that is that one can only evaluate 100 or 200 sperm per sample. The advent of flow cytometry in the early 1970's and the development of new stains has led to more accurate means of assessing sperm viability. Fluorescein diacetate (FDA), carboxyl-fluorescein diacetate (CFDA) in combination with propidium iodide (PI) were applied to flow cytometric assessment in the early 1980's. With flow cytometry several thousands of sperm can be analyzed quickly and without detriment. Staining sperm with FDA, CFDA and PI is useful but is time dependent leading to deterioration of the sample which can alter results. Hoechst stain, 33342 and 33258 were classed as vital stains in 1979. Hoechst 33342, a highly permeable fluorescent stain became prominent for use to differentiate X from Y sperm based on binding in a stoichiometric manner to DNA. Hoechst 33258, a useful exclusion dye, will only stain sperm with lysed membranes. More recently a new membrane permeant dye, SYBR-14 has been used in combination with PI and is characterized by entering only those cells possessing a membrane potential. It shows a very high correlation with living sperm and a highly negative correlation with PI stained dead sperm. Initial studies show that the SYBR-14/PI combination is time insensitive, which allows one to evaluate semen outside of a strict time regimen. The use of CTC and FITC-PSA to ascertain the condition of the acrosome, using both the fluorescent microscopy and flow cytometry has proven to be a very useful tool. Finding one single test to evaluate the fertilizing potential of sperm continues to be elusive. However, using several tests, in combination, such as motility, acrosomal integrity, and a fluorescent assessment of membrane integrity would add significant credibility to estimating sperm function.

Until recently, attempts to preserve porcine embryos have been unsuccessful. Vitrification has been developed as a method of cryopreserving mammalian embryos by avoiding ice crystal formation, assuring a cryopreserved glass state during storage in liquid nitrogen. Vitrification may be a useful method of overcoming the deleterious effects of chilling injury when pig embryos are cryopreserved using conventional slow freezing procedures. In this study, we applied vitrification procedures for rodent and/or bovine embryos to cryopreserve porcine embryos. Following warming, survival was defined as normal development of embryos in culture, namely the formation or reexpansion of the blastocoelic cavity. Experiment 1 tested the relative toxicity of 3 vitrification procedures on Day-5, 6 and 7 porcine embryos. Embryos equilibrated in vitrification solution (VS3a) continued to develop in vitro at rates comparable to that of untreated control embryos. Experiment 2 was designed to evaluate embryonic development following cryopreservation by vitrification in VS3a. Day-5 porcine embryos did not survive cryopreservation while Day-6 and Day-7 embryos survived and continued development in vitro. In Experiment 3, we evaluated a period of culture prior to vitrification and its effect on cryosurvivability of porcine embryos. A 3-h culture period prior to vitrification had no effect on cryosurvivability over that of freshly recovered, immediately vitrified embryos. These studies indicate, for the first time, that porcine embryos can be successfully cryopreserved by vitrification based on morphology and subsequent development in vitro. However, survival following cryopreservation appears to depend upon embryonic age or stage of development.

The effects of luteinizing hormone (LH) (0, 100, 10,000 lU/ml) and follicle-stimulating hormone (FSH) (20 μg/ml) supplementation during in vitro maturation of slaughterhouse-derived oocytes on polar body formation and embryo development subsequent to in vitro fertilization and nuclear transfer were evaluated. Go-nadotropin supplementation of maturation medium in the presence of serum neither enhanced the proportion of oocytes forming a polar body nor significantly affected development following in vitro fertilization or nuclear transfer, except at the highest LH concentration. A very high concentration of LH (10,000 lU/ml) significantly decreased polar body formation, initial cleavage, and blastocyst development (P < 0.05). © 1993 Wiley-Liss, Inc.

Follicle stimulating hormone (FSH) is a glycoprotein hormone with a short half-life and has to be given twice daily for 3-4 days to induce superovulation in heifers. Since such a regimen is time consuming we compared the ovulatory response and yield of embryos in heifers following superovulation with either once or twice daily injections of pFSH for 4 days during the mid-luteal phase of a synchronized estrous cycle or during a prolonged luteal phase in heifers which had been immunized against prostaglandin F2alpha (PG). In Experiment 1, crossbred heifers (n = 42) previously actively immunized against a PG immunogen were superovulated in a 2 (cyclic or persistent corpus luteum) x 2 (once or twice daily injection) factorial plan. The heifers were superovulated with 75 units pFSH, which was injected subcutaneously once (22.5, 22.5, 15 and 15 units per day) or twice daily (9.3 units per injection) for 4 days. In Experiment 2, cyclic crossbred beef heifers (n = 80) were superovulated using pFSH which was given randomly to heifers once daily subcutaneously (T1) or twice daily intramuscularly (T2) using the same daily dose of 9, 7, 5, and 3 mg per day. Estrus was induced in all heifers in both experiments using 500 mug and 250 mug Cloprostenol 12 hours apart on the third day of pFSH injections. All heifers were inseminated twice with frozen-thawed semen at 12 and 24 hours after the onset of standing estrus or at 56 and 72 hours after the first PG if estrus was not observed. Embryos were recovered at slaughter and graded on a scale of 1 to 5 (1 = excellent, 5 = degenerated). Data were recorded for the number of corpora lutea (CL), large (>/=10 mm) and medium (5-9 mm) follicles, number of embryos recovered and embryo morphology. Data were analyzed by least squares analysis of variance procedures. In Experiment 1, there was no difference in ovulation rate between main effects. Fewer embryos were recovered from heifers with a persistent corpus luteum (pCL) and injected once daily (1.71+/-.75 vs 5.75+/-1.27) than from any other group. Heifers with pCL yielded lower (P < 0.05) numbers of freezable embryos than cyclic animals, regardless of injection regimen. In Experiment 2, T2 heifers had a significantly higher number of CL (16.4+/-1.7 vs 7.7+/-1.7; P = 0.0003), large follicles (4.1+/-0.5 vs 2.8+/-0.5; P = 0.04), medium follicles (6.4+/-0.7 vs 4.4+/-0.7; P = 0.04), embryos recovered (9.6+/-1.1 vs 4.9+/-1.1; P = 0.0025) and freezable embryos (4.7+/-0.7 vs 2.1+/-0.7; P = 0.014) than T1 heifers. It is concluded that a single daily subcutaneous injection of pFSH results in a lower superovulatory response than the twice daily regimen in heifers.

It is estimated that as many as 30,000 offspring, mostly cattle, have been produced in the past 5 years using AI or some other means of transport with spermatozoa sexed by flow cytometric sperm sorting and DNA as the marker of differentiation. It is well documented that the only marker in sperm that can be effectively used for the separation of X- and Y-chromosome bearing spermatozoa is DNA. The method, as it is currently used worldwide, is commonly known as the Beltsville Sperm Sexing Technology. The method is based on the separation of sperm using flow cytometric sorting to sort fluorescently (Hoechst 33342) labeled sperm based on their relative content of DNA within each population of X- and Y-spermatozoa. Currently, sperm can be produced routinely at a rate of 15 million X- and an equal number of Y-sperm per hour. The technology is being applied in livestock, laboratory animals, and zoo animals; and in humans with a success rate of 90-95% in shifting the sex ratio of offspring. Delivery of sexed sperm to the site of fertilization varies with species. Conventional AI, intrauterine insemination, intra-tubal insemination, IVF with embryo transfer and deep intrauterine insemination are effectively used to obtain pregnancies dependent on species. Although sperm of all species can be sorted with high purity, achieving pregnancies with the low numbers of sperm needed for commercial application remains particularly elusive in swine. Deep intrauterine insemination with 50-100 million sexed boar sperm per AI has given encouragement to the view that insemination with one-fiftieth of the standard insemination number will be sufficient to achieve pregnancies with sexed sperm when specialized catheters are used. Catheter design, volume of inseminate, number of sexed sperm are areas where further development is needed before routine inseminations with sexed sperm can be conducted in swine. Cryopreservation of sex-sorted sperm has been routinely applied in cattle. Although piglets have been born from frozen sex-sorted boar sperm, freezing and processing protocols in combination with sex-sorted sperm are not yet optimal for routine use. This review will discuss the most recent results and advances in sex-sorting swine sperm with emphasis on what developments must take place for the sexing technology to be applied in commercial practice.

Conceptus loss during the preimplantation and early postimplantation period hinders the efficiency of swine reproduction. Significant conceptus loss occurs during trophectoderm elongation between gestational day 11 ( D11) and day 12 ( D12). Elongation of the porcine conceptus is a key stage of development during which maternal recognition of pregnancy, initial placental development, and preparation for implantation occurs. The objective of this study was to establish comparative transcriptome profiles of D11 ovoid and D12 filamentous conceptuses and thereby identify temporally regulated genes essential for developmental progression during conceptus elongation. Serial analysis of gene expression (SAGE) libraries were constructed from in vivo derived ovoid and filamentous swine conceptuses to yield a total of 42,389 tags (ovoid) and 42,391 tags (filamentous) representing 14,464 and 13,098 putative unique transcripts, respectively. Statistical analysis of tag frequencies revealed the differential expression of 431 tags between libraries ( P < 0.05). Nucleotide sequence alignment searches on public databases provided SAGE tag annotation and gene ontology assignments. Comparisons between the SAGE profiles of ovoid and filamentous conceptuses revealed increased expression of key genes in the steroidogenesis [cytochrome P-450scc ( CYP11A1), aromatase ( CYP19A), and steroidogenic acute regulatory protein ( STAR)] and oxidative stress response pathways [microsomal glutathione S-transferase 1 ( MGST1) and copper-zinc superoxide dismutase ( SOD1)]. Differential expression of these genes in the steroidogenic and oxidative stress response pathways was confirmed by real-time PCR. These results validate the utility of SAGE in the pig and establish an initial model linking gene expression profiles at the pathway level with phenotypic progression from ovoid to filamentous stages of conceptus development.

The objective of this study was to evaluate the in vitro and in vivo developmental competence of parthenogenetic (parthenote) pig embryos derived from ovulated and in vitro matured (IVM) oocytes. A total of four experiments were carried out. These demonstrated that the mean blastocyst rates from stimulated ovulated and IVM pig oocytes were not significantly different (61% vs. 46%, p > 0.05) following in vitro culture. Both ovulated and IVM pig parthenotes were able to develop in vivo for 30 days. Parthenote fetuses collected 21 and 30 days post estrus were morphologically normal but significantly smaller and lighter than fertilized controls (p < 0.01). IVM pig parthenotes stopped development around 31 days post estrus.

The development of embryo freezing technologies revolutionized cattle breeding. Since then, advancements in cryobiology, cell biology, and domestic animal embryology have enabled the development of embryo preservation methodologies for our other domestic animal species, including sheep and goats. Recently, technologies have been developed to cryopreserve pig embryos, notorious for their extreme sensitivity to cooling; horse embryo cryopreservation is in its infancy. While cryopreservation can enhance the utilization of in vitro embryo production technologies, cryosurvival of in vitro-produced (IVP) or micromanipulated embryos is less than that of in vivo-derived embryos. This review outlines recent efforts in livestock embryo cryopreservation. In the near future, use of preserved embryos could be a routine breeding alternative for all livestock producers providing 1) preservation methods for maternal germplasm, 2) global genetic transport, 3) increased selection pressure within herds, 4) breeding line regeneration or proliferation, and 5) methodology for genetic rescue.

Since the development of embryo freezing technologies for cattle in the 1980s, advances in cryobiology, cell biology and embryology of domestic animals have enabled the development of embryo preservation methodology for the pig, notorious for extreme sensitivity to cooling. This review outlines recent efforts to understand the biology of pig embryos as related to their extreme sensitivity to cooling. Cellular analyses and molecular approaches are discussed that have enabled pig embryos to survive cryopreservation and after transfer develop into live offspring with normal fecundity at maturity. In the near future, use of preserved embryos will be a routine breeding alternative for swine producers, providing: preservation methods for maternal germplasm; global genetic transport; increased selection pressure within herds; breeding line regeneration or proliferation; and methodology for genetic resource rescue. It took almost 50 years after the first successful embryo transfer to develop embryo preservation in the pig. Nonetheless, by applying novel methods described herein, rapid progress has been achieved.

The utilization of in vitro produced pig embryos for commercial production or research is dependent upon the development of improved methodology. Our objective was to establish a consistent in vitro embryo production (IVP) system and subsequently utilize the procedures to evaluate culture system components and boar effects. To summarize the IVP system, 403 inseminated oocytes from a total of 2243 were analyzed across 17 replicates for maturation and fertilization efficiency, while 1838 zygotes were cultured in 26 replicates for developmental data. Penetration, cleavage and blastocyst development rates were determined at 18, 44 and either 144 or 168 h post insemination, respectively. Monospermic penetration averaged 31.8+/-7.3% while polyspermy was 30.8+/-17.2%. Cleavage rate was 44.9+/-16.1%, with 21.8+/-7.5% of fertilized oocytes and 51.9+/-15.9% of cleaved embryos developing to blastocysts. For culture medium comparison, fertilized oocytes were cultured in either BECM-6, BECM-7, NCSU-23 or NCSU-23aa and supplemented on Day 5 post insemination (pi) with 10% FCS. These treatments resulted in 4.0, 4.9, 19.8 and 13.6% (+/-3.2%) blastocysts by Day 7 pi, with an average cell number of 44.4+/-9.0, 65.1+/-8.2, 61.3+/-4.5 and 64.4+/-4.8, respectively. These IVP procedures consistently produced zygotes from semen of several different boars, capable of forming blastocysts in vitro. Comparison of developmental rates among the boars indicated that this system is variable among boars but not strictly boar-dependent. Culture media comparisons suggest that NCSU-23 yielded a higher percentage of blastocysts than the other media in this IVP system.

The objectives for the present experiments were to apply sperm sexing technology to an in vitro production system with porcine oocytes obtained from slaughterhouse material. On six experimental days, ovaries were obtained from an abattoir, and cumulus-oocyte-complexes were matured in vitro. Semen was collected from mature boars of proven fertility and was sorted for X-chromosome-bearing sperm, using the Beltsville Sperm Sexing Technology incorporating the use of high-speed sorting. A total of 5,378 oocytes were submitted for in vitro fertilization (IVF). Of these, 559 ova were stained for cytogenetic analysis 18 h after IVF. From the remaining 4,819 ova, 1,595 cleaved, and 1,300 of the cleaved embryos were transferred into 26 synchronized recipients (5 control gilts for unsorted sperm, 21 gilts for X-sorted sperm). In a test of two fertilization media (FERT-A vs FERT-B) higher cleavage rates (P

In vivo-matured porcine oocytes were fertilized in vitro with X and Y chromosome-bearing spermatozoa, and sorted for sex on the basis of DNA content by flow cytometry. Developmental competence of the sexed embryos was determined through established pregnancies after embryo transfer. Spermatozoa were stained with Hoechst 33342 and sorted using a flow cytometry cell sorter. Purity of sorting was 83% for Y spermatozoa and 92% for X spermatozoa. A total of 387 mature cumulus-oocyte-complexes (COC) was collected from 18 superovulated prepuberal gilts shortly before ovulation. In vitro fertilization with sorted spermatozoa was performed in 4 replicates. After 18 h of sperm- oocyte co-culture at 39 degrees C, the zygotes were placed into culture medium (NCSU-23) for another 24 h. The average cleavage rate was 56.2%. Ninety-two embryos produced from X-sorted sperm cells were transferred surgically into the uterus of 2 recipients. Two gilts farrowed and delivered 6 and 4 healthy female piglets, respectively. Additionally, 2 gilts were inseminated intratubally via surgical laparotomy with either X or Y sorted spermatozoa (2 x 10(5)) per oviduct. The 2 sows farrowed producing 15 piglets. Thirteen of the 15 piglets were of the predicted gender (85%).

The objective of these experiments was to determine the efficacy of the new membrane permeant nucleic acid stain, SYBR-14, for assessing boar sperm viability and to determine it's effect on fertilization and early embryonic development using the pig as a model. We examined the staining patterns of SYBR-14 and another vital stain, Hoechst 33342, both in combination with the dead cell stain, propidium iodide (PI), to quantify the proportion of living and dead spermatozoa in ejaculated and epididymal semen. Flow cytometry analyses of semen from 4 boars revealed significant differences among boars for the proportion of SYBR-14-stained spermatozoa in both epididymal and ejaculated samples, but not for Hoechst 33342 or PI stained spermatozoa. Gilts were inseminated with unstained spermatozoa or spermatozoa stained with 2 levels of SYBR-14 or 2 levels of the reference stain, Hoechst 33342. Embryos recovered at 42 to 48 h postinsemination were morphologically evaluated, and only 4 to 8-cell embryos were continued in culture. Overall, fluorescent staining of boar spermatozoa with SYBR-14 or Hoechst 33342 neither affected their ability to fertilize oocytes, nor the developmental competence of the resultant embryos.

Background The domestic pig is an important livestock species and there is strong interest in the factors that affect the development of viable embryos and offspring in this species. A limited understanding of the molecular mechanisms involved in early embryonic development has inhibited our ability to fully elucidate these factors. Next generation deep sequencing and microarray technologies are powerful tools for delineation of molecular pathways involved in the developing embryo. Results Here we present the development of a porcine-embryo-specific microarray platform created from a large expressed sequence tag (EST) analysis generated by Roche/454 next-generation sequencing of cDNAs constructed from critical stages of in vivo or in vitro porcine preimplantation embryos. Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using 454 Titanium pyrosequencing technology. Over one million high-quality EST sequences were obtained and used to develop the EMbryogene Porcine Version 1 (EMPV1) microarray composed of 43,795 probes. Based on an initial probe sequence annotation, the EMPV1 features 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA, 4,121 splice variants in 2,862 genes and a total of 12,324 Novel Transcript Regions (NTR). After re-annotation, the total unique genes increased from 11,961 to 16,281 and 1.9% of them belonged to a large olfactory receptor (OR) gene family. Quality control on the EMPV1 was performed and revealed an even distribution of ten clusters of spiked-in control spots and array to array (dye-swap) correlation was 0.97. Conclusions Using next-generation deep sequencing we have produced a large EST dataset to allow for the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo-specific array was confirmed with a high-level of reproducibility using current Agilent microarray technology. With more than an estimated 20,000 unique genes represented on the EMPV1, this platform will provide the foundation for future research into the in vivo and in vitro factors that affect the viability of porcine embryos, as well as the effects of these factors on the live offspring that result from these embryos.

Swine production requires a stable health status that can be compromised by introduction of live animals for genetic change. Our objective was to use embryo transfer to avoid disease transmission during genetic relocation. Forty genotype-specific (GS) donor females were scheduled for 3 sessions of embryo recovery at 6-week intervals using Altrenogest (Matrix®, Intervet, Millsboro, DE), 1250 IU of equine chorionic gonadotropin (eCG/PMSG; Sigma, St. Louis, MO) and 750 IU of human chorionic gonadotropin (hCG; Chorulon®, Intervet). Single-sire GS matings were made 34 h after Chorulon® injection. To accomplish single-sire transfers, color specific (CS) supplemental embryos were used to assist in maintenance of recipient pregnancy. The CS embryo donors and GS embryo recipients were synchronized with Matrix®, P.G. 600® (200 IU hCG, 400 IU PMSG, Intervet) and Chorulon®. Embryos from GS donors were surgically recovered on Day 5 post-insemination, washed per IETS recommendations using a zwitterion-buffered culture medium (PorcPro E-Blast, Minitube of America, Verona, WI) and transported in a portable incubator (Minitube of America) 2.5 h to the recipient herd. Embryos were surgically transferred into –24-h asynchronous recipients within 6 to 14 h after recovery. A total of 620 embryos were recovered from 65.2% (60/92) of GS matings, and 587 (59.4%) GS and 402 (40.6%) CS embryos were transferred into 63 recipients. On average, 9.3 GS and 6.4 CS embryos were transferred per recipient (15.7). A total of 33 GS embryos were discarded before transfer. To achieve a target of 17 embryos per transfer, 59 embryo transfers required CS embryos and 4 embryo transfers were only GS embryos. Fifty-three (84.1%) recipients were confirmed pregnant by ultrasound at 35 days of gestation. Of the 40 GS donors, one was culled for genetic reasons, 6 did not give transferable embryos, and 1 gave transferable embryos but the corresponding recipient returned to estrus for a total genetic transfer rate of 80% (32/40). After 3 sessions of embryo transfer, 32/33 (97%) GS donors that produced embryos for transfer were represented by a minimum of 1 pregnant recipient at 35 days of gestation; 17/32 GS donors were represented by a single pregnancy and 15/32 by multiple pregnancies. Sera from GS donors were evaluated for porcine reproductive and respiratory syndrome (PRRSV) and porcine circovirus type 2 (PCV2) before each embryo recovery session. Serology results were negative for PRRS (0/98)) and positive for PCV2 in 27.5% (27/98) of GS donors. Embryo wash media from the last 2 washes from PCV2 positive GS donors producing embryos for transfer were pooled and evaluated for PCV2 after transfer; all samples (0/18) were negative for PCV2. In summary, zona pellucida-intact embryos were successfully used to relocate swine genetics from a donor herd into a recipient herd with no apparent health status change in the recipient herd.

In an effort to optimize the number of live offspring from cloned and transgenic pig embryos, embryos are surgically transferred into the isthmus region of the oviduct soon after micromanipulation. Surgical embryo transfer in pigs is successful but still an invasive process. Laparoscopic embryo transfer (Besenfelder et al. 1997 Theriogenology 47, 1051–1060) is much less invasive than surgery and is more adaptable to a variety of commercial embryo transfer conditions. Our goal was to develop the use the laparoscope as an alternative method of embryo transfer for micromanipulated embryos. Naturally cycling maternal white line donor and recipient females were used for laparoscopic embryo transfer. Donors were selected to be in estrus 0 to 24 h before recipients. Two- to four-cell embryos were surgically recovered via mid-ventral laparotomy and immediately prepared for laparoscopic transfer into the oviduct through the infundibulum or through puncture of the oviduct into the ampulla. Sixty-nine embryos (range, 13–20) were transferred into the oviduct via the infundibulum of four recipients. Two recipients farrowed (50%), one recipient spontaneously aborted on Day 27, and one returned to estrus on Day 25 of the estrous cycle. Twenty-one pigs were born (10.5 pigs/farrowed sow) resulting in 30% (21/69) of transferred embryos becoming live offspring. Ninety-seven embryos (range, 10-21) were transferred into the ampulla of the oviduct of six recipients. Four recipients farrowed (67%) and 2 returned to estrus on Day 20 and Day 28. Thirty-nine pigs were born (9.5/sow farrowed) resulting in 39% (38/97) of transferred embryos becoming live offspring. Observations indicate that it is much easier to find the oviduct and deliver embryos via puncture into the ampulla than find and insert the transfer catheter into the infundibulum. Using the efficiencies generated from these data, 1.1 more pigs can be expected per transfer by transferring embryos to the oviduct via puncture than into the oviduct via the infundibulum. These data demonstrate the effective use of the laparoscope for oviductal embryo transfer. Further work is needed to determine if the laparoscope improves the production of live offspring compared to surgical embryo transfer.

The motility, viability (percent live), capacitation status and in vitro fertility of boar spermatozoa were examined, after staining with Hoechst 33342 and flow cytometric sorting in the absence or presence of seminal plasma. Viability was higher in unstained controls and when seminal plasma was present in the medium used to collect spermatozoa from the cell sorter than when seminal plasma was absent or in the staining extender only, but motility was highest when seminal plasma was included in the extender only, compared with the controls and other treatments. The proportions of capacitated spermatozoa were increased by sorting, but were lower when seminal plasma was present, rather than absent, from the staining extender and the collection medium. Compared with unstained controls, extension and staining without sorting only increased the proportion of capacitated spermatozoa after washing in preparation for in vitro fertilization. The percentages of polyspermic, penetrated and cleaved oocytes were lower when inseminated with unsorted (stained) than control (unstained) spermatozoa, regardless of the presence or absence of seminal plasma. These parameters were higher for sorted than for control spermatozoa in the absence of seminal plasma, but in its presence penetration and cleavage were substantially lower. The proportions of capacitated spermatozoa were lower when seminal plasma was present in the collection medium only than in the staining extender or when it was absent altogether, but the former treatment substantially reduced the proportions of polyspermic, penetrated and cleaved oocytes, and the proportion of blastocysts. These findings indicate that sperm capacitation associated with flow cytometric sorting can be reduced by the inclusion of seminal plasma in the collection medium, but this treatment reduces the ability of spermatozoa to fertilize oocytes in vitro under these conditions.