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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.

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.

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 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.