bluuebunny/crossref_metadata_2025 huggingface dataset:
354 EFFECT OF OOCYTE-SECRETED FACTORS (OSF) ON EMBRYO DEVELOPMENT OF OOCYTES FROM SMALL FOLLICLES OF PREPUBERTAL GOATS
Authors: Romaguera R. , Jiménez-Macedo A. R. , Izquierdo D. , Morató R. , Català M. , Roure M. , Palomo M. J. , Paramio M. T.
Published: August 2010

Prepubertal goat ovaries contain a great number of small follicles, between 2.5 and 3 mm in diameter (Martino A et al. 1994 Theriogenology 41, 969-980). Oocyte-secreted factors (OSFs) act on granulosa cells to perform multiple functions required for an appropriate development of the oocyte. In bovines, co-culture of COCs with denuded oocytes (DOs) during IVM improved COCs embryo development dueto OSFs secreted byDOs (Hussein T et al. 2006 Dev Biol. 296, 514-521). In each step of follicle growth, the OSFs secreted are different (Eppig JJ et al. 2001 Reproduction. 122, 829-838). Our hypothesis is that oocytes from small follicles could improve their embryo development by co-culturing with DOs which secrete additional OSFs. Oocytes from prepubertal goats were classified according to their follicular origin: large follicles (>3 mm), small follicles (<3 mm), and control (oocytes recovered by slicing technique and selected by their morphology). The COCs from small follicles were divided in 3 groups and each one transferred into 150-μL droplets of IVM (TCM-199 with serum, hormones and cysteamine): 1) 30 COCs alone (small group); 2) 30 COCs co-cultured with 75 denuded oocytes from small follicles (SDO group); 3) 30 COCs co-cultured with 75 denuded oocytes from large follicles (LDO group). After 27 h of IVM, COCs were fertilized in vitro and the presumptive zygotes were cultured for 8 days in SOF with 10% FCS. Two samples of oocytes were used as control groups: oocytes fertilized (IVF group) and oocytes activated (activated group).At 48 h and 8 days post-insemination (pi), respectively, cleavage and blastocyst rates were recorded. Results are shown in Table 1. Differences between treatment groups were assessed using Fisher’s exact test (Graph-Pad software, San Diego, CA, USA). Values with P < 0.05 were considered statistically significant. Oocytes from small follicle had a significantly lower cleavage and blastocyst rates than IVF and activation oocyte groups. The co-cultured of COCs with SDOs and LDOs increased significantly the blastocyst rate compared to COCs from small follicles. In conclusion, additional OSFs secreted by DOs, improved blastocyst yield of oocytes coming from small follicles. However, not differences were found between OSFs secreted by small or large follicles of prepubertal goat oocytes.

Table 1.
363 ENHANCEMENT OF COMMERCIAL BOVINE IN VITRO EMBRYO PRODUCTION BY SEXING FROZEN SEMEN PRIOR TO INSEMINATION
Authors: Ardais D. B. , Yamazaki L. T. S. , Landim Junior L. P. , Benzi E. C. D. , Corneglian D. P. , Romano M. , Castro Netto A. , Guidorizzi F. , Santos G. L. , Yamazaki W.
Published: August 2010

The high commercial demand on production of genetically valuable female cattle was emphasized after use of sex-sorted semen in commercial in vitro embryo production (IVEP) programs and a reduction of interest in the use of embryo biopsy followed by PCR. Brazil is a leader in IVEP of bovine embryos, and frozen-thawed, sex-sorted samples become a new option for embryo sexing. There are only a few reports in the literature using this new technique for IVEP. Most reports have involved the use of frozen-thawed then sorted for use with AI in Europe. In the present study, frozen semen from three Nelore bulls was used and the blastocyst production rate was evaluated at Day 7 of IVP and compared to previous data from the same bulls and a batch of non-sorted semen. Cumulus oocyte complexes obtained from high genetic merit donors by ovum pickup were matured (TCM-199, supplemented with FCS, LH, FSH, estradiol, pyruvate, and antibiotics) for 24 h and fertilized (Fert-TALP supplemented with BSA, phenylalanine, and heparin) for 18-22 h (Day 0) in vitro. Frozen semen straws were thawed at 35°C for 30 s in a water bath and then selected by centrifugation at 800 g on discontinuous Percoll™ gradients (45 :90%). Samples of frozen-thawed sex-sorted semen were processed from straws from bulls that in some cases may already be dead. Frequently, 3 to 5 straws were thawed, flow-sorted, and then centrifuged to provide sufficient sperm to fertilize 100 oocytes. On Day 1, presumptive zygotes were transferred to culture media (SOFaa supplemented with BSA and FCS) and on Day 7 blastocyst production rate was evaluated. The present results were commercially satisfactory because the frozen-thawed sex-sorted semen from all three bulls performed comparably and, in one case, better than non-sorted semen from the same bulls. Therefore, its use is extremely attractive in commercial bovine IVEP systems and is a more efficient system than embryo sexing.

Table 1.Embryo production rates at 7 days post insemination
369 EFFECT OF HOLDING TIME OF RAW HOLSTEIN FRIESIAN BULL EJACULATES BEFORE SEXING ON NON-RETURN RATES AFTER 56 DAYS
Authors: Knijn H. M. , Frijters A. C. J. , Roelfzema J. E. , Bakker M. , Lenz R. , Merton J. S.
Published: August 2010

Flow cytometric seperation of X- and Y-chromosme-bearing sperm is the only reliable technique that has been used succesfully for commercialization of sexed semen in the dairy industry. However, sperm sexing results in lower fertility compared to conventional semen. Due to logistics like the distance between bull station and sorting laboratory, the number of flow cytometers, and the time needed for the sorting process itself, the holding time between ejaculation and freezing of sex-sorted semen is variable. This holding time could influence the fertility of sex-sorted semen. The aim of this study was to evaluate the effect of different holding times before sexing on the non-return rates on Day 56 post insemination (NRR56). Following collection, raw ejaculates were transported at room temperature (19°C) from two semen collection centers to the central sorting laboratory. Within 2-4 h after collection, volume, sperm concentration and quality of the ejaculates were determined. When accepted, aliquots of 1 mL of semen were stained every 45-60 min for 2 h. Subsequently, sexing was performed according to procedures described previously, using MoFlo SX™ sperm sorters (Garner DL and Seidel GE 2008 Theriogenology 69 : 886-895). Each aliquot of 1 mL semen was sorted for 45-60 min. Four flow sorters were used, so not more than 4 aliquots could be processed simultaneously. After sorting 3-4 h, semen was pooled and a charge number was assigned to this fraction of the ejaculate (called a freezerun). This procedure was done a maximum of 3 times. The time between collection of the raw ejaculate and freezing of the semen was 7-10 h for freezerun 1, 10-14 h for freezerun 2, and 13-18 h for freezerun 3. From September 2007 to April 2009, a total of 16,523 inseminations with sexed semen from 43 different Holstein bulls were performed for which non-return rates at 56 days (NRR 56) were recorded. There were no significant differences in NRR56 between the 3 freezeruns (t-test) (Table 1). The results shown in this study suggest that raw Holstein semen can be held at room temperature for up to 13-18 h prior to sexing and freezing without an effect on NRR56. This study did not indicate what the maximum holding time prior to sexing and freezing would be before resulting in a fertility decline.

Table 1.Number of records and NRR56 for different freezeruns
373 STRATEGIES TO IMPROVE CONCEPTION RATES IN DAIRY HEIFERS INDENTIFIED IN ESTRUS USING TAIL HEAD PAINTING AND INSEMINATED WITH SEXED SEMEN
Authors: Sá Filho M. F. , Ayres H. , Fosado M. , Campos Filho E. P. , Baruselli P. S. , Miguel J. A.
Published: August 2010

Two strategies to improve the conception rates in dairy heifers identified in estrus using tail head painting and inseminated with sexed semen were evaluated. In the first trial, the effect of GnRH injection (100 μg of gonadorelin) at estrus on ovarian responses and conception rates (CR) were verified. A total of 413 virgin Jersey heifers 11 to 12 mo of age were used. All heifers were treated with 25 mg of dinoprost tromethamine at the first day of the AI program. All inseminations were performed 12 h after estrus detection using sexed semen (2.1 million sperm) and the estrus was detected based upon removal of tail-head paint. At estrus detection, heifers were randomly assigned to receive GnRH or not (GnRH = 212 and No GnRH = 201). A subset of heifers (GnRH = 70 and No GnRH = 70), was scanned by ultrasound every 12 h after estrus detection until ovulation. The largest follicle diameter at estrus (LF), ovulatory follicular diameter (OF), ovulation rate (OR), interval between beginning of estrus and ovulation (IEO), and the proportion of heifers presenting late ovulation (LO, ovulation occurred with more than 36 h after the beginning of estrus) were recorded. The GnRH did not influence the LF (13.3 ± 0.9 v. 13.4 ± 0.2 mm; P = 0.87), the OF (13.4 ± 0.3 v. 13.9 ± 0.2 mm; P = 0.17), or OR [98.6% (69/70) v. 97.1% (68/70); P = 0.57]. However, GnRH treatment tended to shorten the IEO (29.2 ± 1.1 h v. 32.0 ± 1.3 h; P = 0.07) and reduced the occurrence of LO (2.9% (2/69) v. 16.2% (11/68); P = 0.02). However, GnRH injection did not influence the CR [GnRH = 47.2% (100/212) v. No GnRH = 51.7% (104/201); P = 0.38]. The second trial evaluated the CRin dairy heifers after the use of single or double insemination dose. A total of 576 Jersey heifers receiving first, second, or third insemination services were used. The heifers were assigned according to sire utilized into one of the three groups (Single 12 h, Double 12 h, or Double 12/24 h). In Single 12 h (n = 193) or Double 12 h (n = 193) groups, heifers received one or two insemination doses (2.1 million sperm) of sexed semen 12 h after estrus detection, respectively. The Double 12/24 h (n = 190) group heifers received two doses 12 h apart from each other (first dose at 12 h and second dose at 24 h after estrus detection). Differences were found between the number of AI services (P = 0.004) on the CR [First = 55.3%a (115/208); Second = 46.1%a (94/204); Third = 34.8% (57/165); P = 0.004]. However, there was no effect of treatment on the CR [Single 12 h = 45.1% (87/193); Double 12 h = 44.0% (85/193); Double 12/24 h = 49.5% (94/190); P = 0.51]. In conclusion, the GnRH injection altered the timing of ovulation, but failed to increase the CR. Furthermore, double insemination doses of sexed semen did not influence the CR in dairy heifers.

Dalhart Jersey Ranch-TX, USA and Sexing Technologies-SP, Brazil.
374 SEX-DEPENDENT METABOLIC DIFFERENCES OF BOVINE PREIMPLANTATION EMBRYOS
Authors: Sturmey R. G. , Bermejo-Alvarez P. , Gutierrez-Adan A. , Rizos D. , Leese H. J. , Lonergan P.
Published: August 2010

Sex-dependent differences in mammalian embryo phenotype are apparent at the preimplantation stage of development, before the appearance of sex-specific cells. The ratio of male:female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling (AAP), a defined, non-invasive metabolic marker of developmental potential to compare the effect of sex on the metabolism of bovine preimplantation blastocysts and expanded blastocysts conceived in vivo (n = 35) or produced in vitro (n = 172). Blastocysts were incubated individually for 24 h in synthetic oviduct fluid medium plus a close-to-physiological mixture of amino acids. The depletion or appearance of 18 amino acids was measured using high-performance liquid chromatography. Blastocysts were then sexed by PCR and the outcome related to AAP. Amino acid depletion by in vitro-produced blastocysts was higher than in embryos conceived in vivo (P = 0.02). Net appearance of amino acids was higher in the medium from early blastocysts produced in vitro (P = 0.018) although this rise was lost at the expanded stage. There were marked differences in the amino acid profiles of male and female embryos produced in vitro: female embryos exhibited significantly increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro-produced blastocysts exhibited gender-specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo-produced blastocysts exhibited differences in 2 out of 18 amino acids. These differences had disappeared by the expanded blastocyst stage. Our experiments reveal striking differences in the metabolism of preimplantation embryos conceived in vivo and in vitro, some of which, particularly in the case of the in vitro-produced embryos, are dependent on embryo sex. Moreover, in vivo-derived embryos tend to have a reduced metabolism consistent with the Quiet Embryo Hypothesis, which proposes that higher quality embryos have less molecular and cellular damage than those of a lower quality and thus have a reduced need to take up nutrients for repair processes.

Supported by a Wellcome-VIP/University of York Fellowship to RGS.
378 INVOLVEMENT OF THE SPERM TAIL IN TRIGGERING OOCYTE ACTIVATION IN PIGS
Authors: Nakai M. , Ito J. , Sato K. , Noguchi J. , Kaneko H. , Kashiwzaki N. , Kikuchi K.
Published: August 2010

In mammals, repetitive increases of the intracellular Ca2+ level, known as Ca2+ oscillations, are observed in oocytes immediately after sperm-oocyte fusion, which is a prerequisite event for oocyte activation. Previous studies indicate that phospholipase C zeta (PLCζ), a strong candidate sperm factor for triggering Ca2+ oscillations, is localized in the sperm head of several mammalian species. We have reported that the rate of pronucleus formation in oocytes injected with a sperm head is lower than that for oocytes injected with a whole spermatozoon (Nakai et al. 2009 IETS). This has given rise to a hypothesis that not only the sperm head but also the tail play a role in inducing oocyte activation in pigs. In this study, we attempted to detect the localization of PLCζ in the pig sperm tail and also its ability to activate porcine oocytes after injection. To clarify the localization of PLCζ in pig sperm, frozen-thawed ejaculated pig sperm were immunostained using an anti-PLCζ antibody that has been reported previously (Kurokawa et al. 2005). Western blotting was also carried out to examine whether PLCζ (72 kDa) was present in the sperm tail. Sperm tails were detached from the head by sonication and then collected after centrifugation in a Percoll density gradient. We also confirmed whether the sperm tail itself had the ability to trigger oocyte activation using the following 4 injection groups: (1)1 sperm head (Head), (2) 1 sperm tail (Tail), (3) 1 sperm head and 1 tail (Head + Tail), and (4) Sham. The nuclear status of the injected oocyte was evaluated at 10 h after injection. In the present study, we used 3 sperm samples that were prepared from different boars. In pig sperm, the acrosome, tail, and post-acrosomal regions were stained by the PLCζ antibody. The signals in both the post-acrosomal and tail regions disappeared after pretreatment with antigenic peptide, but that in the acrosome region was retained. Furthermore, we confirmed the presence of a band of approximately 72 kDa from the sperm tail and also confirmed its disappearance upon pretreatment with antigenic peptide. The rates of oocytes released from metaphase-II arrest in the Head, Tail, and Head+Tail groups were significantly higher than that in the Sham group (P < 0.05 by ANOVA andTukey test). However, most of the oocytes in the Tail group failed to form pronuclei and showed other meiotic stages (anaphase-II, telophase-II, or metaphase-III). In conclusion, we have shown that PLCζ is expressed in the post-acrosomal and tail region of pig sperm. It is suggested that, in the pig, the sperm tail participates in the triggering of oocyte activation.

The authors thankRafaelA. Fissore (Department ofVeterinary and Animal Sciences, University of Massachusetts Amherst) for providing the antigenic peptide for PLCζ. This study was supported in part by JSPS Fellowship (71310042 to M.N.) from the Japanese Society for Promotion of Science (JSPS).
381 LABELING AND ANALYSIS OF SWINE ADIPOSE-DERIVED STEM CELLS WITH CARBOXYFLUORESCEIN DIACETATE AND QUANTUM DOTS
Authors: Cieslak N. , Massie A. , Wilson S. M. , Monaco E. , Wheeler M. B.
Published: August 2010

The quantity, accessibility, and abundance of subcutaneous adipose tissue in humans make it an attractive alternative to bone marrow as a source of adult stem cells for therapeutic purposes. Adult adipose-derived mesenchymal stem cells can differentiate into a variety of lineages including adipose, bone, cartilage, and muscle. In addition, the use of adult stem cells for regenerative medicine rather than those from embryos avoids concerns with ethics, safety, and immunology. One important issue is the ability to track the transplanted stem cells during the regeneration process to evaluate the stem cell-mediated healing. The objective of this study was to compare the efficiency, longevity, and intensity of carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and quantum dot nanocrystal (Qtracker™, Invitrogen, Carlsbad, CA, USA) labeled adipose-derived stem cells (ADSC) over an in vitro culture period of 4 weeks. Adipose-derived stem cells (6 x 106) previously isolated and frozen at -196°C were thawed and cultured in 75-cm3 flasks with 14 mL of DMEM. Cells were grown to 80% confluence and trypsinized. After trypsinization, the cells were divided into 4 treatments (3 x 106 cells per treatment). The treatments were (1) unlabeled control, (2) labeled with 30 μM CFDA SE, (3) labeled with 15 nM Qtracker™, and (4) labeled with 15 nM Qtracker™, following the Invitrogen Qtracker™ protocol. Cells (1 x 106) were removed from each treatment every week for 4 weeks and fixed in formalin for later analysis. When all the samples were collected, they were analyzed using flow cytometry. Data were analyzed via chi-square test. The percentage of cells labeled with CFDA SE and Qtracker™ was 99.35 and 98.46%, respectively, immediately after labeling. By 1 wk, the percentage of cells labeled with CFDA SE and Qtracker™ had deceased (P < 0.01) to 0.11 and 1.48%, respectively. The CFDA SE-labeled cell percentages had decreased (P < 0.01) to 0% at 2, 3, and 4 wk, respectively. The Qtracker™-labeled cells also decreased (P < 0.01) to 0.745, 1.69 and 0.45% at 2, 3, and 4 wk, respectively. The high rate of cell division of these cells in vitro might be responsible for the rapid loss of both labels during the first week of culture. Previous results from our lab have shown that the CFDA SE is retained in the cells for up to 6 wk in vivo (Lima AS et al. 2006 Reprod. Fertil. Dev. 18, 208). Similar studies need to be done with the quantum dot-labeled cells to determine the Qtracker™ label’s longevity in vivo. In conclusion, quantum dots can be used to label ADSC, in vitro, for at least 4 wk, albeit at much lower levels than those observed during the week following labeling. Determination of a suitable label for high-percentage porcine ADSC labeling during long-term in vitro culture remains to be completed.

This research was supported by the Intel Scholar’s Program and the Illinois Regenerative Medicine Institute.
385 ESTROGENIC EVALUATION OF ALKYPHENOLS IN MOUSE EMBRYONIC STEM CELLS
Authors: Jung E. M. , Jeung E. B.
Published: August 2010

Xenoestrogens can have adverse effects on the reproductive and immune systems; for example, 4-tert-octylphenol (OP) and 4-nonylphenol (NP) can have estrogenic effects in target cells. In this study, we investigated the effects of xenoestrogens on the expression of undifferentiation and differentiation markers in mouse embryonic stem (ES) cells, which are important mediators of the differentiation of ES cells into cardiomyocytes. The ES cells were treated with 17β-estradiol or OP and NP in a time-dependent manner (for 1, 2, or 3 days), and embryoid bodies (EB) cells were given the same treatment for 5, 8, 12, or 16 days. The mRNA expressions of undifferentiation markers (Oct-4, Sox2, Zfp206, and Rex-1) and cardiomyocyte differentiation markers (α-MHC, β-MHC, ANF, and MLC-2V) were determined by semi- and quantitative real-time PCR. Treatment with E2 induced an increase (1.3- to 4.6-fold) in Oct-4 expression at the transcriptional level in a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2, Zfp206, or Rex-1 genes in ES cells, suggesting that E2 might be an Oct-4 enhancer in ES cells. However, induction of Oct-4 expression by E2 might result from changes in the Oct-4 promoter methylation pattern rather than from other regulatory mechanisms. We also found that cardiomyocyte differentiation markers were differentially expressed in response to xenoestrogens in EB cells. Taken together, these results suggest that xenoestrogens might play a role as a positive regulator of the undifferentiation process in mouse ES and EB cells and might be involved in the maintenance and differentiation of mouse ES cells.
386 HORSE AMNION: A SOURCE OF MESENCHYMAL (AMSC) AND EPITHELIAL STEM CELLS
Authors: Lange Consiglio A. , Corradetti B. , Bizzaro D. , Cassano M. , Cremonesi F.
Published: August 2010

According to the developmental stage from which they are obtained, stem cells are classified as being embryonic, fetal, or adult. Embryonic stem cells have unlimited self-renewing capacity and multilineage differentiation potential, but the separation of these cells requires destruction of the embryo. Moreover, their clinical application seems to be hindered by the high tumorigenic rate after transplantation. Stem cells derived from adult tissues are considered to be more limited in their potential; although, they are currently the more versatile cells in the clinical field. However, the risk of the immunological rejection of the transplanted stem cells by the recipient is an important limiting factor. In human medicine, stem cells isolated from term placenta are the ideal candidates for disease treatment, specifically because of their plasticity and reduced immunogenicity. The aim of this work was to provide, for the first time, an isolation protocol and the characteristics of the stem cells from horse amniotic membrane, which hold potential uses in equine clinical regenerative medicine. Minimal criteria for stemness definition are adherence to plastic culture dish, formation of fibroblast colony forming units (CFU-F), specific pattern of surface antigen expression, and differentiation potential toward one or more lineages. The amnion is a thin, avascular membrane composed of an epithelial layer and an outer layer of connective tissue. From 3 samples of allantoamnion retrieved at delivery, each amniotic membrane was stripped from the overlying allantois and, for isolation of the epithelial cells, digested with trypsin. After removal of epithelial cells, the AMSC population was obtained by digestion with collagenase and DNase. The cellular yield from term amnion was 10-fold more epithelial cells than AMSC. Isolated cells readily attached to plastic culture dishes. Culture was established in DMEM-HG medium, supplemented with 10% serum and EGF, where the cells proliferated robustly. Epithelial cells displayed typical cuboidal morphology, whereas AMSC were fibroblast-like. Normally, 5 to 6 passages were achieved before proliferation decreased, with a mean of 13.08 and 26.5 cell population doublings after 31 days, respectively, for epithelial cells and AMSC. The mean frequency of CFU-F was, respectively, 1 : 283 and 1:111 for epithelial cells and AMSC. The 2 cellular lines expressed MSC mRNA markers (CD29, CD105, CD44) and were negative for CD34, which was expressed at the fifth passage in both cellular types. Osteogenic differentiation of epithelial stem cells and AMSC was confirmed by von Kossa stain and by an increased expression of osteocalcin and osteopontin. Our preliminary data showed that equine amnion holds apparent potential as a source of presumptive stem cells, which might have widespread clinical applications, but aspects including immunohistochemical study, preclinical experimentation, and immunological properties must be studied.
39 EFFECT OF TIME OF FIXED-TIME AI ON PREGNANCY RATES IN ZEBU CROSS-BRED BEEF HEIFERS TREATED WITH PROGESTERONE RELEASING DEVICES AND ESTRADIOL CYPIONATE
Authors: Ramos M. , Cutaia L. , Chesta P. , Bó G. A.
Published: August 2010

Two experiments were designed to evaluate the effect of the timing of fixed-time AI (FTAI) in relation to the removal of an intravaginal progesterone-releasing device (1 g of progesterone, DIB, Syntex SA, Buenos Aires, Argentina) on pregnancy rates in Bos indicus × Bos taurus cross-bred heifers. In experiment 1, 285 Bonsmara × zebu cross-bred heifers, between 18 and 24 months of age and with a body condition score (BCS) between 3.0 and 3.5 (1-5 scale) were used. On the day of initiation of treatment (Day 0), the heifers’ ovaries were palpated (92% of them had a CL) and they received a new DIB plus 2 mg of estradiol benzoate (EB; Syntex SA) and 250 μg of cloprostenol (Ciclase DL, Syntex SA). On Day 8, DIB devices were removed and all heifers received 250 μg of Ciclase plus 0.5 mg of estradiol cypionate (ECP; Cipiosyn, Syntex SA). At that time the heifers were randomly divided to receive FTAI between 48 to 49 h, 53 to 54 h, or 58 to 59 h after DIB removal. The heifers underwent FTAI with semen from 4 bulls by 2 inseminators. In experiment 2, 260 heifers from the same group as those used in experiment 1 (87% with a CL) were treated exactly as those in experiment 1, except that previously used DIB was inserted on Day 0. Pregnancy diagnosis was performed 30 days post-fixed-time AI by ultrasonography. The data were analyzed by logistic regression, taking into account the effect of time of FTAI, semen, and inseminator on pregnancy rates. In experiment 1, pregnancy rates were lower (P = 0.04) in the heifers undergoing FTAI between 48 and 49 h after DIB removal (46/95, 48.4%) than those undergoing FTAI 53 to 54 h (61/99, 61.6%) or 58 to 60 h (57/91, 62.6%) after DIB removal. However, no differences in pregnancy rates were found (P = 0.72) in experiment 2 between the 3 treatment groups, with 39/91 (42.9%) for the 48 to 49 h group, 45/89 (50.6%) for the 53 to 54 h group, and 35/89 (43.8%) for the 58 to 59 h group. There was no effect of the semen or inseminator (P > 0.2) in either experiment. We conclude that when Bos indicus × Bos taurus beef heifers are synchronized with new DIB devices and ECP, higher pregnancy rates are obtained in heifers undergoing FTAI late (between 53 to 60 h after DIB removal) than in those undergoing FTAI early (48 to 49 h after DIB removal). However, time of insemination does not apparently affect pregnancy rates when Bos indicus × Bos taurus beef heifers are synchronized with previously used DIB devices and ECP.
391 A COMPARATIVE GENE EXPRESSION ANALYSIS BETWEEN SHEEP EARLY EMBRYONIC DEVELOPMENT AND PUTATIVE EMBRYONIC STEM CELLS
Authors: Murray B. M. , Schmoelzl S. , Andronicos N. M. , Hill J. R. , Verma P. J. , Holland M. K.
Published: August 2010

The optimization of culture media to support the isolation of embryonic stem cells relies on methods to monitor whether the pluripotent state of the cultured cells has been maintained. We developed a panel of gene expression assays that allowed us to correlate molecular measures of pluripotency or lineage differentiation with a developmental time course. By conducting quantitative PCR analysis of sheep embryos over Day 6.5 to 24 and sheep inner cell mass (ICM) cells cultured over 25 days, we tested whether culture media designed to inhibit differentiation are able to maintain sheep ICM cells in a pluripotent state. Briefly, embryos were collected from Merino ewes (n = 50, 3 years) at Day 6.5, 12, 16, 20, and 24 post-AI. Embryos were collected from the dissected uterine tracts of slaughtered ewes, excluding Day 6.5 blastocysts, which were surgically recovered from superovulated ewes. For the in vitro culture, Day 6.5 ICM cells were isolated by immunosurgery and cultured on mitomycin-C-treated mouse embryonic fibroblasts in an inhibitor-based medium (3i, based on Ying Q-L et al. 2008 Nature 453, 519-523). Real-time PCR assays for pluripotency (OCT4, SOX2, NANOG) and differentiation (ectodermal: FGF5, PAX6; endodermal: GATA4, GATA6, Somatostatin; mesodermal: BMP4, Connexin40) of sheep candidate genes were conducted on cDNA prepared from these samples and normalized against the reference genes RPL19 and RPS26. In in vivo embryos, pluripotency markers OCT4, SOX2, and NANOG all decreased between Day 6.5 and Day 20, although OCT4 expression spiked around Day 16. More interestingly, pluripotency expression decreased during in vitro culture, with NANOG expression completely lost by passage 2 at Day 11 and OCT4 expression at an equivalent Day 24 embryo basal level by Day 14. The endodermal markers GATA6 and GATA4 decreased between Day 6.5 and Day 12, respectively, although in vitro GATA4 was only expressed once at Day 7. In vivo FGF5 and both PAX6 and Somatostatin displayed a delayed onset, increasing expression from Day 16 and 20, respectively, whereas the ectodermal markers were already expressed by Day 7 in vitro. Both mesodermal markers Connexin40 and BMP4 presented minor fold changes in both data sets. In conclusion, this study has verified the primer sets and described a sheep in vivo embryo gene expression profile comprising both pluripotent and differentiation candidates. Furthermore, the decrease of pluripotency markers together with the appearance of differentiation markers during in vitro culture of ICM cells suggest that culturing ICM cells in 3i media is not sufficient to maintain a sheep-specific pluripotent population of cells. Therefore, future studies will be aimed at manipulating the current in vitro system to focus on maintaining pluripotent genes such as NANOG and OCT4 in culture.
395 OVINE SPERMATOGONIA ISOLATION AND IN VITRO GROWTH ASSAY
Authors: Qi A. , Wuliji T. , Zhang Y.
Published: August 2010

Spermatogonia, as adult stem cells from the male reproductive system, are attracting strong interest from those studying male reproductive gamete preservation and developing new approaches in transgenic animals. Protein gene product 9.5 (PGP9.5) is a marker of sheep spermatogonia, which has been validated by J. R. Rodriguez-Sosa et al. (2006).The objective of this study was to develop an in vitro culture system for spermatogonial growth. Twenty 2- to 3-month-old rams were randomly selected at a local slaughter house for testicular tissue collection. Two-step enzymatic digestion methods were used for spermatogonia isolation from seminiferous tubules. In brief, mechanically isolated seminiferous tubules from testicular tissue were incubated in 1:1 1 mg mL-1 collagenase and hyaluronidase with 5 μg mL-1 DNase I for 20 min at 37°C. Most of the surrounding interstitial cells will fall off from seminiferous tubules by slightly pipetting. Seminiferous tubules were from cell suspensions after natural sedimentation in PBS and then were digested in 0.25% trypsin + 0.04% EDTAfor 5 to 7 min at 37°C to disassociate tubules into single cells. Data were analyzed with ANOVA procedures. Means of 5 specimens were presented. Of the total isolated cells, 19.7 ± 5.3% were identified as PGP9.5+cells, and 23.8 ± 3.6% were identified as c-kit+ cells. C-kit, the transmembrane tyrosine kinase receptor for stem cell factor, has been identified, which is expressed and functional in differentiating A1-A4 spermatogonia but not in spermatogonial stem cells (OhtaH2000). For in vitro culture of spermatogonia, DMEM supplemented with 1X ITS (insulin, transferrin, selenium), 100 μM β-mercaptoethano, 6 mM L-Glutamine, and 1X nonessential amino acids were used as basic culture medium. We have found that in primary spermatogonia culture, cells cultured together with testis somatic cells (sertoli cells) in basic medium supplied with 2.5% fetal bovine serum (FBS) were much more efficient than culturing with a supplement of 10% FBS. In primary culture, growing round-shaped cell colonies were visible from Day 5 in basic culture medium. In subculture, colonies were enzymically digested into smaller pieces from Day 8 to 10 and then placed onto mouse embryonic fibroblasts feeder layer in 2.5% FBS basic medium with the addition of 100 ng mL-1 glial cell-derived neurotropic factor, 10 ng mL-1 leukemia inhibitory factor, and 10 ng mL-1 basic fibroblast growth factor. PGP9.5+ spermatogonia cell colonies maintained their normal round shape until 4 to 5 passages. However, in subsequent passages, the colonies became flattened and cells gradually lost their interconnection and the growth pattern present in the early passages. Also in subsequent passages, cells began expressing more C-kit than PGP9.5. In conclusion, PGP9.5+ spermatogonia were successfully isolated from 2- to 3-month-old ram testis, and PGP 9.5 cell colonies were maintained and proliferated in the in vitro culture system up to 2 months.
397 DERIVATION OF NEURAL STEM CELLS FROM PORCINE EPIBLAST CELLS
Authors: Rasmussen M. A. , Hall V. J. , Hyttel P.
Published: August 2010

The use of neural stem cells (NSC) has gained increased attention as a means of treating neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. However, before regenerative treatment of humans can be undertaken, safety studies of NSC using animal models are required. The mouse has been the model of choice so far; however, testing in larger mammals such as the pig is essential. The aim of this study was to derive NSC from porcine epiblast cells and to analyze these cells using neural stem cell markers. A total of 47 epiblasts were isolated from E9 porcine embryos and grown on mouse embryonic fibroblast cells in a porcine embryonic stem cell medium. After 5 days, 23 outgrowth colonies had formed (49%). Based on morphology, 8 outgrowth colonies were selected and cut into 63 smaller pieces, which were transferred to MS5 stromal cells in a serum replacement medium, and after an additional 12 days, rosette structures had formed. These structures were transferred to Matrigel-coated dishes in a neural stem cell medium containing EGF and FGF. Under such conditions, bipolar cells containing large nuclei and several nucleoli grew out from the rosettes. The bipolar cells have been expanded for more than 8 passages without any change in morphology or growth rate, and upon high-density culture, the cells spontaneously form floating neurospheres. Stainings revealed that the cells expressed the neural stem cell markers Nestin (100%), Sox2 (100%), Pax6 (100%), and Vimentin (100%), as well as the proliferation marker Ki67 (54%). The same markers were found to be expressed in the lateral ventricles of the developing porcine brain, a location known to have high neurogenic activity. When growth factors were withdrawn from the culture medium, a higher proportion of TujI expressing cells were observed, especially when cells were cultured as neurospheres. We conclude that it is possible to derive presumptive NSC from porcine epiblast cells and that these express the same markers as reported for human NSC. Further studies are required to determine if the cells can be cultured long term and differentiate into various neuronal and glial cell types.
4 CHARACTERIZATION OF LENTIVIRAL SHORT-HAIRPIN RNA EXPRESSION VECTORS CONTAINING SINGLE OR MULTIPLE BOVINE POLYMERASE III PROMOTERS
Authors: Peoples M. , Westhusin M. , Golding M. , Long C.
Published: August 2010

Lentiviral vectors have become an important and efficient molecular biology tool to integrate foreign DNA into target genomes. These vectors have been previously used in our laboratory to make cloned transgenic fetuses expressing short-hairpin RNAs (shRNAs) targeting the caprine prion mRNA (Golding et al. 2006 Proc. Natl. Acad. Sci. USA 103, 5285-5290) and bovine myostatin mRNA. Specially designed shRNAs have a robust ability to decrease protein expression by initiating a mRNA destruction pathway or by translational inhibition. However, initial experiments targeting foot and mouth (FMDV) viral RNA have indicated that polymerase (Pol) II promoters may be unable to produce enough mature shRNA particles to significantly knock down viral replication in vitro. The goal of this research project was to identify and utilize bovine Pol III promoters to express shRNAs in lentiviral vectors and to express multiple unique shRNAs from a single lentiviral vector using different Pol III promoters. This goal is particularly important to the successful reduction of FMDV replication in a cell, as it limits random mutations from escaping the shRNA-mediated viral genome destruction. The 3 bovine Pol III promoters we selected were 7sk, U6-2, and H1. They were individually amplified from the same genomic DNA preparation. The promoters were inserted immediately upstream of our shRNA expression sequence, resulting in lentiviral vectors designated GT-b7sk, GT-bU6-2, and GT-bH1.To confirm that the promoters were functional, a luciferase reporter assay was performed in HEK 293T cells, where each vector expressed either a shRNA targeting luciferase (luc) or a non-specific shRNA.All promoters expressing luc shRNA resulted in significant reduction of luciferase activity between 68 and 80% compared with non-targeting controls. In addition, there was no significant difference between Pol III promoters when analyzing reduced luciferase activity. In the second phase of the study, we developed 7 unique combinations of 2 or 3 Pol III shRNA expression cassettes to test individual shRNA function with one shRNA designed to target luciferase and the others non-targeting. In multiple Pol III expression constructs, the U6-2 and 7sk promoters resulted in the greatest reduction of luciferase activity at 89 and 95%, respectively. In addition, luciferase activity was reduced to the greatest extent when the luc shRNA was expressed from the second (82%) or third (87%) Pol III cassette. Overall, bovine Pol III-based promoters are effective at expressing shRNAs from a lentiviral vector. In addition, multiple Pol III shRNA expression cassettes can be inserted into a single lentiviral vector and still achieve significant reduction of target protein. These vectors will be used to create transgenic cattle and pigs that express multiple shRNAs targeting the FMDV genome with hopes of creating animals that are resistant to FMDV.
40 TIMING OF INSEMINATION WITH SEXED OR NONSEXED SEMEN ON PREGNANCY RATES OF JERSEY HEIFERS DETECTED IN HEAT BY RADIOTELEMETRY
Authors: Sales J. N. S. , Crepaldi G. A. , Fosado M. , Campos Filho E. P. , Baruselli P. S.
Published: August 2010

The objectives of this study were to evaluate the follicular dynamics (experiment 1) and the effects of the timing of insemination with sexed or nonsexed semen on pregnancy rates (experiment 2) of Jersey heifers detected in heat by a radiotelemetric estrus detection system. In experiment 1, 43 Jersey heifers, around 12 mo old and BCS of 2.68 ± 0.11 (1 to 5 scale) were used. The Heat Watch (HW) system was utilized to detect the onset of estrus and mounting behavior associated with estrus. Ultrasound examinations to monitor follicular dynamics occurred every 12 h from estrus onset for 48 h. Statistical analyses were performed using GLM and GLIMMIX procedure of SAS (SAS Institute, Cary, NC, USA). The results of experiment 1 indicated a mean ovulatory follicle diameter of 14.1 ± 0.3 mm, ovulation rate of 86.1% (37/43), and an interval of 31.2 ± 0.9 h from onset of heat to ovulation. In experiment 2, 753 Jersey heifers were allocated in a 2 × 4 factorial with semen (sexed and nonsexed) and AI period (0 to 6, 6 to 12, 12 to 18, and 18 to 24 h after heat onset) as parameters. Semen from 3 bulls was used, with ejaculates divided in 2 fractions: one fraction was submitted to the traditional freezing procedure and the other was submitted to the sexing process and then frozen. The statistical analysis was performed using GLIMMIX procedure of SAS. There was no interaction among the semen, bull, and AI period. There were effects on pregnancy rate by type of semen [sexed 49.5% (189/382) and nonsexed 64.2% (238/371); P = 0.001] and by bull [bull A 53.5% (107/200)b, bull B 50.0% (108/216)b and bull C 63.4% (211/333)a; P = 0.008]. Semen from bull C resulted in a greater pregnancy rate for both sexed and nonsexed semen. Within semen type, there were no differences in pregnancy rates by AI moment [sexed: 0 to 6 h 48.2% (41/85), 6 to 12 h 48.7% (54/111), 12 to 18 h 49.5% (49/99), 18 to 24h 52.4% (44/84) and nonsexed: 0 to 6h 62.8% (49/78), 6 to 12h 60.6% (63/104), 12 to 18h 68.0% (68/100), 18 to 24h 64.8% (57/88); P = 0.77]. We conclude that the use of sexed semen resulted in a lower pregnancy rate than nonsexed semen, and that AI timing does not affect conception rate in Jersey heifers identified in estrus by radiotelemetric estrus detection system. However, there was a bull effect on conception rate.

The authors wish to thank Sexing Technologies and Dalhart Jersey Ranch.
400 KARYOTYPIC ANALYSIS AND EPIGENETIC MODIFICATIONS OF CULTURED PORCINE ADIPOSE TISSUE-DERIVED STEM CELLS
Authors: Williams K. J. , Bondioli K. R. , Godke R. A.
Published: August 2010

The introduction of genetic modifications in donor cells for NT requires a significant number of population doublings (PD), and the deleterious effects, which may be attributed to aneuploidy or changes in DNA methylation and histone acetylation, are difficult at this time to circumvent. We hypothesize that the identification of a donor cell that is genetically stable for a long period of time in vitro such as somatic stem cells or those cells that demonstrate stem-like characteristics may be reprogrammed more completely, thus providing the key to increasing the efficiency of NT. Regulators of development in undifferentiated cells are suggested to be silenced by the presence of a bivalent domain modification pattern in which a large region of repressive histone 3 lysine 27 trimethylation (H3K27me3) contains smaller regions of activating histone 3 lysine 4 trimethylation (H3K4me3).The dual marks work to silence developmental genes in embryonic stem cells while simultaneously keeping them receptive to activation. The objectives of the current study were to determine the chromosomal stability of porcine adipose tissue-derived adult stem cells (pASC) through in vitro culture, to analyze pASC alongside fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins and global methylation and acetylation patterns, and to determine the presence of a co-enrichment of H3K27me3 and H3K4me3 within the promoter regions of developmentally important transcription factors. Metaphase spreads were prepared, and the presence of H3K27me3 and H3K4me3 was investigated in each of 3 individual pASC primary cultures for each analysis; whereas, gene expression and global methylation and acetylation were analyzed in each of 4 individual pASC and FPF primary cultures. Of 714 metaphases analyzed, 509 (71.3%) were aneuploid and only 205 (28.7%) were normal diploid porcine cells. For each cell population, we found a remarkable percentage of aneuploidies (43.7, 48.9, and 47.3, with a 46.6 ± 1.5 average) present immediately after the cultures were established. Chi-square analysis indicated that the percent of aneuploid cells during PD 1-10 was significantly less than that for PD 11-20 and PD 21-30. Also, porcine ASC demonstrated a consistently lower level of DNA methylation and histone acetylation through passages 2 through 7; whereas, the patterns for FPF varied. The expression levels of chromatin remodeling transcripts remained lower in pASC throughout culture when compared with FPF. Finally, porcine ASC possess a co-enrichment of H3K27me3 and H3K4me3 on the promoter region of the developmentally important transcription factor OCT-4. In vitro-cultured porcine ASC used as donor cells for NT should be chosen from early PD because of increased levels of aneuploidy at later PD. With a more complete characterization of porcine ASC, a donor cell population that can be more efficiently reprogrammed following fusion with the oocyte might be identified.
412 OVARIAN RESPONSE IN ALPACAS (VICUGNA PACOS) TO SUPERSTIMULATORY TREATMENT WITH FOLLICLE-STIMULATING HORMONE AND EQUINE CHORIONIC GONADOTROPIN
Authors: Huanca T. , Cardenas O. , Gonzales M. , Huanca W. , Quintela L. A. , Becerra J. J. , Herradon P. G.
Published: August 2010

The objective of this study was to evaluate the ovarian response in alpacas to superstimulatory treatment with FSH and eCG. Alpacas females (n = 49) were examined daily by transrectal ultrasonography using a 7.5-MHz linear-array transducer (Aloka SSD500, Tokyo, Japan) to determine ovarian follicular dynamics. Alpacas (n =41) were selected when a growing follicle ≥7 mm in diameter was detected and were given an i.m. injection of 4 μg of buserelin (Conceptal®, Intervet, Millsboro, MD, USA) on Day 0 (i.e. beginning of treatment). On Day 2, alpacas were assigned to 1 of the following 2 treatments: (1) the FSH group (n = 21), which received a total dose of 200 mg NIH-FSH-P1 of pFSH (Folltropin®-V, Bioniche Animal Health Inc., Belleville, Ontario, Canada) in decreasing dosages of 52, 48, 40, 32, and 28 mg divided in twice-daily i.m. injections for 5 days; and (2) the eCG group (n = 20), which received a single dose of 750 IU of eCG (Folligon®, Intervet, Millsboro, MD, USA). Animals received an i.m. dose of 0.150 mg of triapost (PGF; Iliren, Intervet, Millsboro, MD, USA) on Day 6. On Day 7, ovaries of all alpacas were examined by transrectal ultrasonography, followed by mating and embryo collection on Day 14. Data were analyzed by ANOVA. The mean number of follicles <7 mm in diameter was 5.8 ± 1.0 and 3.2 ± 0.9 for those treated with FSH and eCG, respectively (NS). The number of follicles 7 to 13 mm in diameter was 5.8 ± 1.5 and 9.7 ± 1.2 for the FSH and eCG groups, respectively (P < 0.05). The number of follicles >13 mm in diameter was 0.3 ± 0.4 and 1.0 ± 0.3 for the FSH and eCG groups, respectively (NS). The mean (± SD) follicle diameter was 8.3 ± 0.3 and 9.0 ± 0.2 for the FSH and eCG groups, respectively (NS). The mean (± SD) number of CL was 6.0 ± 1.9 and 8.7 ± 2.3 for the FSH and eCG groups, respectively (NS). Only 82.9% of animals (34/41) were flushed and embryos were recovered in 18 animals (52.9%), with 2.7 ± 0.4 and 2.7 ± 0.7 embryos in the FSH and eCG groups, respectively (NS). Large unovulated follicles (diameter >13 mm) were observed in 34.1% of animals (14/41) on Day 14 and most of them (71.4%) were in the group treated with eCG (10/14; P < 0.05). In conclusion, the use of eCG resulted in a higher number of preovulatory follicles (7-13 mm in diameter) than FSH. However, the percentage of animals with large unovulated follicles was also higher in the eCG-treated group, resulting in no differences in the number of CL counted or embryos obtained per treatment.