bluuebunny/crossref_metadata_2025 huggingface dataset:
133 EFFECTS OF ACTIVINA ON mRNA EXPRESSION IN IN VITRO FERTILIZED BOVINE EMBRYOS CULTURED FROM CHEMICALLY DEFINED TWO-STEP CULTURE MEDIUM
Authors: Park J. E. , Jang G. , Oh H. J. , Hong S. G. , Yang I. S. , Kang S. K. , Lee B. C.
Published: January 2008

During the preimplantation stage, embryo development occurs in a maternal environment within the oviducts and uterine horns. It has been speculated that both the embryo itself and the maternal reproductive tract provide paracrine factors that influence embryo development (Jones et al. 2006 Reproduction 132(5), 799–810). Activins are known for FSH releasers, and several previous studies have reported that activin subunits and activin receptors mRNA were expressed in oocytes, zygotes, and oviduct (Yoshioka et al. 1998 Reprod. Fertil. Dev. 10(3), 293–298; Gandolfi et al. 1995 Mol. Reprod. Dev. 40(3), 286–291). The purposes of the present study were Experiment 1) to evaluate the effects of activin A on developmental competence of bovine embryos derived from two-step defined culture medium (Lim et al. 2007 Theriogenology 67(2), 293–302) and Experiment 2) to analyze the effects of activin A on transcriptional level of the genes in IVF embryos. Cumulus–oocyte complexs were harvested from ovaries obtained from a local slaughter house, matured, and fertilized in vitro. In vitro fertilized zygotes cultured in media supplemented with activin A in the early stage at the concentrations of 0, 10, or 100 ng mL–1 or in the later stage medium at the concentrations of 0, 10, or 100 ng mL–1. Data were analyzed using the Statistical Analysis System (SAS) program. In Exp. 1, although the development competence of embryos that cultured with activin A in the early stage medium was not significantly different, development to blastocysts on day 8 in the later stage medium with 100 ng mL–1 activin A was significantly higher than the control group [22.4% (54/264) v. 34.7% (76/233); P < 0.05]. Hatching rate of blastocyst on day 8 was significantly higher in the presence of 100 ng mL–1 activin A in the later stage culture medium compared with the control group [9.3% (5/54) v. 22.4% (17/76); P < 0.05]. In Exp. 2, the relative expression of 3 genes (Na/KATPase, E-cad, Glut-1) related to blastocyst hatching and implantation was analyzed. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitative reverse transcription-polymerase chain reaction. The expression level of the Na/K ATPase, E-cad, and Glut-1 gene were higher in the presence of activin A in the culture medium compared with the control group. In conclusion, this study suggests that activin A during the later stage of in vitro bovine embryo development can enhance the developmental competence of preimplantation embryos, increase the hatching rate, and affect expression level of genes related to hatching and implantation in defined culture medium.
This study was financially supported by KOSEF (grant ? M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.
134 EFFECT OF ELEVATED CIRCULATING PROGESTERONE CONCENTRATION ON DEVELOPMENT OF IN VITRO PRODUCED BOVINE ZYGOTES IN VIVO
Authors: Rings F. , Carter F. , Hölker M. , Kuzmany A. , Besenfelder U. , Havlicek V. , Tesfaye D. , Schellander K. , Lonergan P.
Published: January 2008

Elevated concentrations of circulating progesterone in the immediate post-conception period have been associated with an increase in embryonic growth rate, interferon-tau production, and pregnancy rate in cattle and sheep. Much of this effect is likely mediated via downstream effects of progesterone-induced changes in gene expression in the tissues of the uterus. However, whether or not progesterone has a direct effect on the embryo also is unknown and, at least in vivo, in single ovulating animals, is difficult to assess. Using state-of-the-art endoscopic techniques, the objective of this study was to examine the effect of elevated progesterone on the development of IVP zygotes transferred to the oviducts of cattle with high or normal circulating progesterone concentrations. Simmental heifers (n = 14) were synchronized using a combination of 2 injections of a prostaglandin F2α analogue administered 11 days apart and gonadotropin-releasing hormone. Only animals exhibiting a clear standing oestrus (= day 0) were used. In order to produce animals with divergent progesterone concentrations, half of the animals received a PRID on day 3 of the oestrous cycle, which was left in place until embryo recovery. All animals were blood sampled daily from days 0 to 7. Cleaved embryos were transferred using endoscopy to the ipsilateral oviduct of each recipient on day 2 and recovered by non-surgically flushing the oviduct and the uterus on day 7. The number of embryos developing to the morula/blastocyst stage was recorded at recovery and following overnight culture in CR1aa medium. Data were analyzed by chi-square analysis. Insertion of a PRID on day 3 resulted in a significant elevation in progesterone concentrations from day 4 (2.36 ± 0.16 ng mL–1 v. 0.54 ± 0.10 ng mL–1, P < 0.001) until day 6 (1.98 ± 0.22 ng mL–1 v. 0.95 ± 0.17 ng mL–1; P < 0.01). The recovery rate was lower in animals that received a PRID (P < 0.05). However, there was no effect of progesterone on the proportion of embryos developing to the morula/blastocyst stage. These results suggest that elevated concentrations of progesterone do not affect the ability of the early embryo to reach the blastocyst stage in vivo and that the reported positive effect of high progesterone levels in terms of fertility are manifested after day 8.

Table 1. Effect of elevated progesterone concentration on development of in vitro produced bovine zygotes in vivo
142 DONOR CONTRIBUTION HAS A LARGER IMPACT ON HSP70.1 EXPRESSION IN BOVINE EMBRYOS THAN THE CUMULUS REMOVAL TECHNIQUE
Authors: Reeder A. L. , Monson R. L. , Watters J. J. , Rutledge J. J.
Published: January 2008

The impact of embryo manipulation and donor contribution on molecular markers was investigated in the bovine model. Transcript expression analysis was performed on Holstein 2-cell embryos 24 h after they underwent one of two cumulus removal techniques. Oocytes from each of 5 abattoir-obtained Holstein ovaries were fertilized with semen of an individual Holstein bull, yielding families of 4, 5, 18, 25, and 28 zygotes. Cumulus cells from zygotes within each family were removed 24 h post-fertilization by either repetitive pipetting with a 135-µm pipette or by manipulation in a microfluidic chamber with a 30-µm aperture. Levels of the stress response transcript, HSP70.1, were used to evaluate cumulus removal technique in the 5 full sib families of developing embryos. Embryonic HSP70.1 cDNA from differentially treated full siblings was analyzed by qPCR and the resultant CT values were normalized to 18S rRNA, the reference gene, to account for potential differences in cDNA levels in each reaction. The data were analyzed by using the GLM procedure in SAS (SAS Institute, Inc., Cary, NC, USA), testing for treatment and family effects. HSP70.1 did not differ between microfluidically treated and pipetted embryos (P = 0.45). However, comparison of HSP70.1 expression across families was different (P < 0.001). These results indicate that pooling of genetically unrelated embryos may skew transcript expression data and influence research conclusions. Additionally, the question of culture conditions on embryo vitality needs to be evaluated without donor health status or heredity being additional factors.
145 REESTABLISHMENT OF AN EXTINCT STRAIN OF SHEEP UTILIZING ASSISTED REPRODUCTIVE TECHNOLOGIES
Authors: Bormann C. , Long C. , Menges S. , Hanna C. , Foxworth G. , Westhusin M. , Pliska V. , Stranzinger G. , Joerg H. , Glimp H. , Millsap L. , Porada C. , Almedia-Porada G. , Kraemer D.
Published: January 2008

The objective of this study was to reestablish an extinct strain of sheep that exhibits spontaneous X-linked factor VIII deficiency closely mimicking human hemophilia A. Twenty female carriers of the trait, produced in a previous study (Bormann et al. 2006 Reprod. Fertil. Dev. 18, 201–202), were backcrossed using 3 straws of semen from their affected sire using either IVF or multiple ovulation embryo transfer (MOET). Eleven oocyte donors were synchronized with CIDRs (15 days) and superovulated with a declining dose of FSH (204 mg) twice daily for 3.5 days. Nine MOET donors were synchronized using CIDRs (14 days), superovulated with a declining dose of FSH (184 mg) BID for 3 days with pregnant mare serum gonadotropin (PMSG; 200 IU) given with the final dose of FSH, and given 1000 IU of hCG 12 h post-CIDR removal. Recipient ewes were synchronized using sponges (Ovakron, HeriotAgvet, Rowville, Victoria, Australia) containing 30 mg of flugestone acetate (14 days) and given PMSG (400 IU) at sponge removal, followed by 1000 IU of hCG 12 h post-sponge removal. Oocytes were collected via follicular aspiration during midventral laparotomy and matured as previously reported. Semen for IVF was prepared by centrifugation on a Percoll gradient. Oocytes and sperm were incubated in mTALP with 20% estrus sheep serum (modified from Bavister et al. 1977 Bio. Reprod. 16, 228–237) for 20 h, then vortexed to remove cumulus cells, and cultured in G1.3 medium (Vitrolife, Englewood, CO) with BSA until transfer. Embryos were surgically transferred into oviducts of recipients 24 to 48 h following IVF. The 9 MOET donors were surgically inseminated at the uterotubal junction with approximately 1–2.0 � 106 spermatazoa. Oviducts of eight of these ewes were flushed 48 h post-insemination with warm M199 containing Hanks salts, 25 mm HEPES, 10% FBS, and 0.5 µg mL–1 gentamicin. MOET embryos were surgically transferred to synchronized recipients within 5 h. One MOET donor was not flushed due to poor response and did not produce an offspring. Utilizing 140 ova, IVF produced 54 embryos for an embryo/oocyte rate of 38.6%. All IVF embryos were transferred into 15 recipients resulting in 3 lambs for a lamb/embryo rate of 5.5%. The MOET donors produced 38 embryos and 13 apparently unfertilized ova, generating an embryo/oocyte rate of 74.5%. MOET embryos were transferred into 21 synchronized recipients. MOET produced 16 lambs for a lamb/embryo rate of 42.1%. Co-transfer of 1 IVF and 1 MOET embryo into a single recipient produced one offspring. Utilizing multiple reproductive technologies over a two-year period, 8 hemophilic offspring (7 females and 1 male), 6 carrier females, and 6 unaffected males were produced. This strain of sheep will be used to produce affected offspring for stem cell-based therapies.
15 HEAT SHOCK COGNATE 70 PROTEIN IMPROVES THE LONG-TERM SURVIVAL OF RAM SPERMATOZOA DURING STORAGE AT 17°C IN A COMMERCIAL EXTENDER
Authors: Lloyd R. E. , Watson P. F. , Holt W. V.
Published: January 2008

Heat shock proteins (HSPs) are highly represented among oviductal epithelial cell (OEC) plasma membrane proteins (Sostaric et al. 2006 J. Proteome Res. 5, 3029–3027); their abundance increases in oviductal fluid in response to the presence of spermatozoa (Georgiou et al. 2005 Mol. Cell. Proteomics 4, 1785–1796), and they are capable of binding directly to spermatozoa (Boilard et al. 2004 Biol. Reprod. 71, 1879–1889). As a result of these observations, a role for HSPs in prolonging the functional life of spermatozoa in the oviduct prior to fertilization has been proposed. Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical AI (Salamon and Maxwell 2000 Anim. Reprod. Sci. 62, 77–111). This is due, in part, to extenders being unable to maintain the functional life of ram spermatozoa to the same extent as the oviduct. Therefore, in this study, we aimed to determine the effect of supplementing extenders with the HSPs, HSC70 and HSP70, on the maintenance of ram sperm function during storage. Eight freshly collected ram semen ejaculates were split and diluted in the extenders, INRA96 and RSD-1, to 25 � 106 spermatozoa mL–1, either alone (control) or supplemented with HSC70, HSP70, or α-tubulin (protein with a molecular weight similar to that of the two HSPs). The final concentration of protein in each case was 4 µg mL–1, and the samples were stored at 17�C for 48 h. At 1 h, 6 h, 24 h, and 48 h, the viability and DNA integrity of the stored spermatozoa was determined using the live and dead stains, SYBR�-14 and ethidium homodimer, and the Sperm-Ovis-Halomax kit, respectively. In each case, the mean percentage (%) of viable and DNA-fragmented (DF) spermatozoa was determined and log-transformed prior to analysis using factorial ANOVA. Statistical significance was defined as P < 0.05. In INRA96, but not RSD-1, the beneficial effect of protein supplementation on the % of viable spermatozoa observed during storage was significant (P < 0.001). Most noteworthy was INRA96 supplemented with HSC70, which maintained the % of viable spermatozoa observed during storage significantly (P < 0.001) better than INRA96 alone. Protein supplementation had no significant effect on the % of DF spermatozoa observed during storage. However, the % of DF spermatozoa observed increased significantly (P < 0.001) during storage, irrespective of the presence or absence of protein. On the basis of these findings, it is not implausible to suggest that supplementing the commercially available INRA96 extender with HSC70 would improve the fertility rates observed following cervical AI using stored ram semen. Furthermore, the increase in the incidence of DNA-fragmented spermatozoa observed during storage offers a potential explanation as to why poor fertility rates are often observed when ram semen stored in conventional extenders is used for cervical AI.
151 EFFECT OF RECIPIENT CATEGORY ON PREGNANCY RATES ON A BOVINE EMBRYO TRANSFER PROGRAM IN PATAGONIA, ARGENTINA
Authors: Villarreal J. , Garcia Guerra A. , Brogliatti G. M.
Published: January 2008

The fertility of the recipient is one of the more important factors conditioning the success of an embryo transfer (ET) program. Selection and management of recipients is a very important contributing factor to achieve high pregnancy rates (Stroud and Hasler 2006 Theriogenology 65, 65–76). From a reproductive point of view, a good recipient is a cow capable of receiving an embryo and taking it to term (Palma et al. 2001 Biotecnologia de la reproduccion, INTA). In beef and dairy cattle, recipients are categorized according to number of births, age, and condition score (Stroud and Hasler 2006). A retrospective analysis was done to confirm a relationship between recipient category, based on their physiologic and reproductive stage, and pregnancy rates. The analysis was done during January and February (2006–2007) based on data recovered from an embryo transfer program done in Chubut province (Patagonia) for the project ‘Polo Genetico Angus’ (Angus Association – Chubut government agreement). Two hundred and seven morulas and/or blastocysts, quality 1 or 2 frozen in 1.5 m ethylene glycol (IETS manual), were transferred randomly in 10 different farms. Embryos were thawed for 10 s at room temperature and 30 s in a water bath at 35°C and transferred by one single technician. Angus, Hereford, or its breeds were used as recipients. A reproductive exam was performed before embryo transfer and, based on it, recipients were classified into 4 categories: heifer (3–4 years old), lactating cow, weaned cow, and dry old cow (Table 1). The synchronization protocol used for recipients consisted of a single administration of D-cloprostenol and heat detection twice a day for 5 days. Data were analyzed by Cochran-Mantel-Haenszel test (chi-square). Results are shown in Table 1. There is a significant difference (P ≤ 0.05) in pregnancy rates among categories. Recipients with higher pregnancy rates were lactating cows and weaned cows 61.6% and 56.9%, respectively, compared to heifers, 45.3%; a lower pregnancy rate was founded in the dry old cows group. Dry cows were animals that failed to get pregnant during the last season. Heifers also were animals that failed the artificial insemination program. In both groups, the reduced fertility may explain their lower pregnancy rates. In conclusion, selection toward fertility of recipients is a very important factor to take into account to design a successful embryo transfer program.

Table 1. Pregnancy rates for recipient categories in an ET program in Chubut (Patagonia), Argentina

This research was supported by Centro Genetico Bovino Eolia.
153 EFFECTS OF OVINE INTERFERON-β ON REPLICATION OF BOVINE VIRAL DIARRHEA VIRUS AND BOVINE HERPESVIRUS-1
Authors: Galik P. K. , Gard J. A. , Spencer T. S. , Marley M. S. , Stringfellow D. A. , Givens M. D. , Edmondson M. A.
Published: January 2008

Bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) are the most commonly isolated viruses from abattoir-origin materials utilized in embryo production and known to associate with zona pellucida-intact (ZP-I) embryos after exposure and washing. Some evidence indicates that developing embryos may produce substances that are able to inhibit viral replication in adjacent cells. Interferons such as recombinant human interferon-α are known to have anti-BVDV activity but no effect against BHV-1. In some preliminary studies, bovine interferon-τ has shown antiviral activities against BVDV but not against BHV-1. However, interferon-τ in other species has not been evaluated for anti-BVDV and anti-BHV-1 effects. Thus, the objective of this study was to evaluate the cytotoxicity and anti-viral effect of ovine interferon-τ against a non-cytopathic high affinity strain of BVDV (SD-1) and BHV-1 (Colorado) in cell culture. Serial dilutions (1:10) beginning with an initial concentration of 1 mg mL–1 of interferon-τ were made in 96-well plates and then Madin Darby bovine kidney (MDBK) cells were seeded in the wells. Cells and interferon-τ were incubated at 37.5�C in 5% CO2 and air for 24 h prior to addition of virus. The following concentrations of BVDV were added to the wells: 6000, 3500, 1000, 625, and 350 cell culture infective doses (CCID50) (50% endpoint) per well. In addition, four viral concentrations of BHV-1, 1000, 500, 250, and 100 CCID50/mL were evaluated in separate cell cultures. Virus isolation was utilized to determine if the addition of interferon-τ decreased the amount of infective virus. Ovine interferon-τ produced no observable cytotoxicity in MDBK cells in any of the assays. Also, the three highest concentrations of interferon-τ significantly decreased the amount of BVDV in all of the concentrations of BVDV tested but had no apparent effect on the concentration of BHV-1 in cell cultures. Therefore ovine interferon-τ has anti-BVDV effects similar to those seen with bovine interferont-τ and neither has any apparent antiviral activity on BHV-1 in cell culture. Additionally, ovine and bovine interferon-τ might serve to limit or prevent the transmission of BVDV and curtail the negative effects of BVDV on oocyte and embryo development. However, a similar effect is not expected for BHV-1.
159 TECHNIQUES FOR OVUM PICK-UP IN GONADOTROPIN-TREATED ALPACAS
Authors: Gamarra G. , Gallegos A. , Alvarado E. , Asparrin M. , Vivanco W.
Published: January 2008

The objective of the present study was to evaluate the quantity and quality of oocytes collected when using 2 methods for ovum pick-up and 2 different regimens for ovarian stimulation in live alpaca donors. Thirty-four non-pregnant female alpacas of 3 to 5 years of age maintained at 4100 m elevation in southern Peru were randomly distributed into 4 experimental groups. Groups 1 (n = 8) and 3 (n = 9) received an intravaginal device containing 0.78 mg of progesterone (Cue-Mate�, Bioniche Animal Health, Belleville, Ontario, Canada) plus an i.m. injection of 1 mg of estradiol benzoate on Day 0; the intravaginal device was removed on Day 7. Groups 2 (n = 7) and 4 (n = 10) received an i.m. injection of 3.1 mg of LH (Lutropin�, Bioniche Animal Health) on Day 0. Females received 700 IU of eCG (Pregnecol�, Bioniche Animal Health) i.m. on Day 7 (Groups 1 and 3) or Day 2 (Groups 2 and 4). In all groups, oocyte collection was done 2 days after the injection of eCG. Groups 1 and 2 were subjected to ventral laparotomy aspirating the oocytes from follicles >3 mm in diameter using a 10-mL hypodermic syringe containing 1 mL of aspiration media (Ringer's lactate solution plus 10% bovine serum) and connected to an 18 G � 1 inch aspiration needle. After collection, the follicular fluid was searched and the COC were graded. Groups 3 and 4 were subjected to ovum pick-up by transvaginal recovery using an ultrasound scanner (Parus 240�, Pie Medical, Maastricht, the Netherlands) equipped with a vaginal probe of 7.5 MHz (MEVA�, Pie Medical) and a 17G � 55 cm aspiration needle introduced through a needle guide. Follicles >3 mm in diameter were aspirated into 50-mL centrifuge tubes containing 5 mL of aspiration media with 75 IU mL–1 of heparin. The aspirated fluid was filtered and rinsed using an embryo filter (EmCon�, Immunosystems, Menomonie, WI), and COC were searched and graded under a microscope based on the intactness of the cumulus cell layers. Data were analyzed by ANOVA. There were no differences (P > 0.05) between groups in the mean number of follicles aspirated per donor (11.0, 13.8, 9.4, and 9.1 for Groups 1 to 4 respectively), and in the mean number of COC recovered per donor (7.6, 7.0, 6.0, and 6.1 respectively for Groups 1 to 4). The proportions of good quality COC were significantly (P < 0.01) different between surgical (81.0 and 79.5% for Groups 1 and 2) and transvaginal/ultrasound-guided (7.4% for Group 3) methods of collection; however, they were similar to the proportion in Group 4 (64.9%) retrievals. The results show that in the absence of an intravaginal device, a similar quantity and quality of alpaca oocytes can be collected when using a surgical approach or minimally invasive ultrasound-guided transvaginal follicular aspiration.
198 FUNCTIONAL EVIDENCE FOR THE EXISTENCE OF AN OVIDUCTAL FACTOR THAT INDUCES ZONA PELLUCIDA HARDENING AND REGULATES POLYSPERMY IN THE PIG AND COW
Authors: Cánovas S. , Grullón L. , Romar R. , Matás C. , Avilés M. , Coy P.
Published: January 2008

Many differences between in vivo and in vitro fertilization (IVF) efficiency in mammals are related to the differences between IVF media and oviductal fluid. One of the best known examples is the frequency of polyspermy observed under in vitro conditions in cattle (Roh S et al. 2002 J. Vet. Med. Sci. 64, 667–671) and, in particular, in pigs (Coy P and Romar R 2002 Reprod. Fertil. Dev. 14, 275–286). Zona pellucida (ZP) resistance to pronase digestion (ZP hardening) has been considered as a postfertilization event contributing to the block of polyspermy in mammals (Green D 1997 Rev. Reprod. 2, 147–156). However, pig and cow unfertilized ovulated oocytes show a ZP hardening of hours or days (Katska L et al. 1999 Reprod. Dom. Anim. 34, 255–259; Kolbe T and Holtz W 2005 Theriogenology 63, 1695–1705) compared with the minutes or seconds observed in the in vitro-matured oocytes, even after fertilization (Coy P et al. 2002 Reproduction 124, 279–288; Coy P et al. 2005 Reproduction 129, 19–26). Consequently, we propose the existence of an oviductal factor that induces ZP hardening before any contact of the oocyte with the sperm, thus regulating polyspermy. Porcine and bovine oviductal fluid was obtained by aspiration of oviducts collected at the slaughterhouse and stored frozen. In vitro-matured porcine and bovine oocytes were incubated for 30 min in the oviductal fluid, washed thoroughly in fresh medium, and either assessed for ZP digestion time or in vitro fertilized. The results, analyzed by ANOVA, showed a very strong ZP hardening in oviductal-treated oocytes (2866.83 � 94.4 s in the pig and 4301.1 � 441.7 s in the cow) compared with control oocytes (63.5 � 2.9 s in the pig and 124.2 � 5.9 s in the cow). Moreover, the percentage of monospermy for the oviductal-treated oocytes was significantly higher in both species (50.0 � 10.0% in the pig and 91.7 � 3.0% in the cow) compared with the control groups (5.56 � 3.8% in the pig and 80.8 � 3.5% in the cow). Percentage penetration did not change in porcine oocytes but decreased in bovine oocytes (58.1 � 3.3 v. 38.4 � 3.3, P ≤ 0.001), whereas the mean number of sperm per oocyte decreased for the porcine-treated oocytes (2.7 � 0.2 v. 8.2 � 0.4, P ≤ 0.001) and did not change for the bovine oocytes. These results support the hypothesis that an oviductal factor induces ZP hardening, contributing to the control of polyspermy in the pig and cow, and that it could be used to improve the output of IVF.
Supported by MEC and FEDER (AGL2006-03495).
206 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON IN VITRO EMBRYO PRODUCTION
Authors: Imai K. , Inaba Y. , Yoshioka H. , Aikawa Y. , Ohtake M. , Suzuki M. , Kobayashi S.
Published: January 2008

We previously reported that follicular wave synchronization, by removal of the dominant follicle on Day 5 after ovum pickup (OPU), was effective in increasing oocyte quality in the developing follicles (Imai et al. 2006 32th Annual Conference of the IETS, poster presentation no. 277). The current study was designed to examine the effect of superstimulatory treatment to induce subsequent follicular wave synchronization on embryo production by OPU and IVM-IVF-IVC in Holstein dry cows. Cows were reared under the same feeding and environmental conditions, and 2 OPU sessions were conducted in each cow. In the first session, OPU was performed in 8 cows on arbitrary days of the estrous cycle by using a 7.5-MHz linear transducer with needle (Cova needle, Misawa Medical, Tokyo, Japan) connected to an ultrasound scanner (SSD-1200, Aloka, Tokyo, Japan). Follicles larger than 8 mm in diameter were then aspirated and a CIDR was inserted on Day 5 (the day of first OPU session = Day 0). Cows then received 30 mg of FSH (Antrin-R10; Kawasaki Mitaka Pharmaceutical Co., Tokyo, Japan) twice a day from Days 7 to 10 in decreasing doses (6, 6, 4, 4, 3, 3, 2, 2 mg) by i.m. injection. Cloprostenol (PGF; Clopromate C; Sumitomo Pharmaceuticals Co., Tokyo, Japan; 0.75 mg) was administered in the morning of Day 9 (third day of superstimulation). The second OPU session was performed 48 h after PGF administration (Day 11), and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Collected oocytes were evaluated by their cumulus cell morphology, cytoplasmic color, and density. Grades 1 and 2 COC were matured, fertilized, and cultured as described by Imai et al. [2006 J. Reprod. Dev. 52(Suppl.), S19–S29]. Embryo development was assessed by the cleavage rate on Day 2 and by the blastocyst formation rate on Days 7 to 8 (the day of insemination = Day 0). Data were analyzed by Student's t-test. There were no differences in the mean (� SD) number of aspirated follicles or collected oocytes between the first (32.5 � 6.8 and 26.0 � 12.7, respectively) and second (29.3 � 10.4 and 19.0 � 9.4, respectively) OPU sessions (P > 0.1). The percentage of Grade 1 and 2 oocytes for the second OPU session (90.5 � 13.8%) was significantly higher (P < 0.01) than for the first OPU session (63.1 � 6.3%), and significant differences were found for cleavage (79.4 � 14.1, 61.8 � 25.1, P < 0.01) and blastocyst rates (68.1 � 16.7, 24.2 � 22.3, P < 0.001) between sessions. The mean numbers of blastocysts obtained per session were 4.3 � 2.9 and 12.8 � 8.7 in the first and second sessions, respectively (P < 0.01). These results indicate that superstimulatory treatment and subsequent follicular wave synchronization were effective on in vitro embryo production by increasing the oocyte quality.
208 EFFECT OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR ON BLASTOCYST DEVELOPMENT AND POST-TRANSFER SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Loureiro B. , Bonilla L. , Entrican G. , Hansen P. J.
Published: January 2008

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that has been implicated in preimplantation embryo development. Granulocyte-macrophage-CSF improves the proportion of bovine embryos that become blastocysts in vitro (Moraes and Hansen 1997 Biol. Reprod. 57, 1060–1065) and increases blastocyst cell numbers in mice (Robertson et al. 2001 Biol. Reprod. 64, 1206–1215). The long-term goal of the present research was to evaluate the effects of GM-CSF on post-transfer survival of bovine embryos. The experiments used recombinant ovine GM-CSF produced in transfected Chinese hamster ovary (CHO) cells or an equivalent volume of cytokine-free CHO cell supernatant (control). The objective of the first study was to evaluate the effects of GM-CSF on post-transfer survival. Embryos were cultured with 10 ng mL–1 of either GM-CSF or cytokine-free CHO cell supernatant added to culture medium at Day 1 after insemination. Embryos were transferred at Day 7 to lactating dairy cows according to a timed embryo transfer protocol. Pregnancy was evaluated at approximately Day 45 of gestation. There was no significant difference in the proportion of embryos becoming blastocysts at Day 7 after insemination (34.8 v. 37.5% for the control and GM-CSF; SEM = 2.4%). There was also no difference in pregnancy rates between cows receiving control embryos (6/24; 25%) and cows receiving embryos treated with GM-CSF (8/35; 23%). A second study determined the effects of various concentrations of GM-CSF on the development of in vitro-produced embryos to the blastocyst stage. Embryos were cultured in 5% (v/v) oxygen (low oxygen) or atmospheric oxygen (21%, w/v; high oxygen) in the presence of 0, 1, 10, or 100 ng mL–1 of GM-CSF or an equivalent volume of cytokine-free CHO cell supernatant (control). The GM-CSF was added on either Day 1 or Day 5 after insemination. Cleavage rate was accessed on Day 3 after insemination. Stage of development was recorded at Day 7 and Day 8 after insemination. There was no effect of GM-CSF on cleavage rate. Addition of GM-CSF at Day 5 to embryos cultured in low or high oxygen increased the percentage of oocytes that became blastocysts at Day 7 (P < 0.01) and Day 8 (P < 0.01), but addition at Day 1 did not have a significant effect on blastocyst development. The greatest effects of GM-CSF occurred at a concentration of 10 ng mL–1. At this concentration, least squares means for the percentage of oocytes that became blastocysts at Day 7 were 13.9 v. 21.6% (control v. GM-CSF) when GM-CSF was added at Day 5, and 19.5 v. 21.5% when GM-CSF was added at Day 1. The percentage of blastocysts at Day 8 was 20.9 v. 28.7% when GM-CSF was added at Day 5, and 26.7 v. 27.5% when GM-CSF was added at Day 1. In conclusion, GM-CSF can affect the competence of embryos to develop to the blastocyst stage, but at the concentrations and times given, there was no evidence that GM-CSF enhanced embryo survival after transfer.
209 PRODUCTION OF IN VITRO-FERTILIZED INTERSPECIES BLASTOCYSTS BETWEEN SHEEP OOCYTES AND GOAT SPERMATOZOA
Authors: Malakar D. , De A. K. , Akshey Y. S.
Published: January 2008

In rodents, chimeric blastocysts produced by combining embryonic cells of 2 different species have been used to investigate cell lineage and cell interaction during development. Interspecific chimerism offers new approaches to the study of reproductive incompatibilities between species. The aim of the present study was to produce interspecies embryos between sheep oocytes and goat spermatozoa through in vitro fertilization. Sheep ovaries were collected from a nearby abattoir and transported to the laboratory in 0.9% normal sterile saline containing antibiotics (50 μg mL–1 of gentamicin sulfate) at 30 to 35°C. Oocytes were aspirated by the puncturing method in a medium consisting of TCM-199 and 3 mg mL–1 of BSA. Only A and B grade COC with 3 or more layers of cumulus cells with homogeneous ooplasm were taken for maturation. The oocytes were washed 4 to 5 times in maturation medium containing TCM-199 (HEPES modified), 10 μg mL–1 of LH, 5 μg mL–1 of FSH, 1 μg mL–1 of estradiol-17β, 50 μg mL–1 of sodium pyruvate, 5.5 mg mL–1 of glucose, 3.5 μg mL–1 of L-glutamine, 50 μg mL–1 of gentamicin, 3 mg mL–1 of BSA, and 10% EGS (heat-inactivated goat serum). The COC (15 to 20 oocytes) were placed in 100-μL droplets of maturation medium, covered with paraffin oil in a 35-mm Petri dish, and incubated in a CO2 incubator (5% CO2 in air) with maximum humidity at 38.5°C for 24 h. Fresh semen was collected from a proven buck. The semen was washed at 300g 2 times in sperm-TALP (Parrish et al. 1986) medium to remove the seminal plasma and incubated with fert-TALP medium containing sperm-TALP supplemented with 50 μg mL–1 of heparin and 3 mg mL–1 of BSA for 1.5 h for capacitation. The matured sheep oocytes with expanded cumulus cells were coincubated with capacitated buck spermatozoa at a concentration of 2 × 106 sperm mL–1 for 10 h in 5% CO2 in air with maximum humidity at 38.5°C. The presumptive zygotes were then cultured in embryo development medium containing TCM-199 (HEPES modified), 0.03 mg mL–1 of sodium pyruvate, 0.1 mg mL–1 of L-glutamine, 0.05 mg mL–1 of gentamicin, 10 μL mL–1 of essential amino acids, 5 μL mL–1 of nonessential amino acids, 10 mg mL–1 of BSA (fraction V), 10% fetal calf serum, and 50 mm cysteamine along with sheep oviductal cells for further development. The cleavage was recorded at 36 to 48 h postinsemination, and morula- and blastocyst-stage embryos were obtained on Day 5 and Day 7, respectively. The cleavage percentage was found to be 58.6%. Among the cleaved embryos, 43% reached the morula stage, and among morula, 31% reached the blastocyst stage. We concluded that interspecies embryos between sheep and goat can be produced successfully in vitro up to the blastocyst stage.

Table 1. In vitro production of different stages of interspecies (sheep × goat) embryos
217 EFFECT OF PRE-EQUILIBRATION TIME ON THE SURVIVAL RATE OF MATURED BOVINE OOCYTES AFTER VITRIFICATION AND ON SUBSEQUENT EMBRYO DEVELOPMENT
Authors: Sugulle A. H. , Dochi O. , Koyama H.
Published: January 2008

Prolonged exposure of oocytes to cryoprotectants causes cell injury, whereas a short exposure time results in insufficient permeation because of ice formation. This study was designed to determine the optimal pre-equilibration time and its effect on the survival rate of matured bovine oocytes after warming and on subsequent embryo development. Bovine COC were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH at 38.5°C in 5% CO2 in air. Then, the COC were partially denuded. The oocytes were pre-equilibrated in 100 μL of vitrification solution 1 (VS1) containing 7.5% ethylene glycol (EG), 7.5% DMSO, and 20% CS in TCM-199 for 0, 1, 3, and 5 min. Then, the oocytes were moved through 100-μL drops of vitrification solution 2 (VS2) containing 30% EG, 30% DMSO, 0.5 m sucrose (Suc), and 20% CS in TCM-199 for 30 s, loaded into cryotops, and immersed into liquid nitrogen. Oocytes were warmed by plunging the cryotops into 1 m Suc in TCM-199 supplemented with 20% CS for 1 min, placed in 0.5 m Suc in TCM-199 supplemented with 20% CS for 3 min, and finally in TCM-199 supplemented with 20% CS alone for 5 min. Frozen–thawed semen from a single bull (5 × 106 spermatozoa mL–1) was used for fertilization. Zygotes were vortexed to remove the cumulus cells 18 h after fertilization and cultured in CR1aa for 9 days. Data were analyzed by the chi-square test. Results are presented in Table 1. There were no differences in the survival rates of the control and vitrified oocytes. The cleavage rate of controls at both 24 and 48 h was greater (P < 0.01) than that of vitrified oocytes. Among pre-equilibration times, the cleavage rate of 0, 1, and 3 min pre-equilibrations at both 24 and 48 h was greater than with the pre-equilibration time of 5 min (P < 0.01). With respect to blastocyst development, the control oocytes showed greater development rates than the vitrified oocytes (P < 0.01), whereas the development rates were lower with the pre-equilibration time of 5 min (P < 0.01) than with the other pre-equilibration times. In conclusion, the results indicated that matured bovine oocytes could survive after vitrification and subsequently develop into blastocysts after IVF. However, the pre-equilibration time before vitrification affects cleavage and blastocyst development; a longer exposure time resulted in lower blastocyst development.

Table 1. Effect of pre-equilibration time on matured bovine oocytes survival and on subsequent embryo development
231 STAGE-DEPENDENT CHANGES OF IP3R1 PHOSPHORYLATION DURING IN VITRO MATURATION AND DEVELOPMENT IN PIG OOCYTES
Authors: Ito J. , Suzukamo C. , Mochida T. , Furugaichi A. , Nakajima N. , Fissore R. A. , Kashiwazak N.
Published: January 2008

During fertilization in mammalian species, a sperm-induced intracellular Ca2+ signal [Ca2+] is well suited to mediate the highly specialized spatiotemporal patterns of [Ca2+]i responses that underlie fertilization. Recently, we demonstrated that the expression pattern of inositol 1,4,5- triphosphate receptor type 1 (IP3R1) did not change during in vitro maturation and parthenogenetic activation in mouse oocytes; however, the phosphorylation status of IP3R1 depended on the cell cycle during meiosis. Moreover, it was shown that IP3R1 phosphorylation played a crucial role in the induction of [Ca2+]i oscillations (Lee et al. 2006 Development 133, 4355–4365). In other species, expression of IP3R1, especially phosphorylation levels of IP3R1 during meiosis, has not been examined. The aim of this study was to examine the kinetics of IP3R1 expression and phosphorylation during in vitro maturation and activation in pig oocytes. Immature oocytes at the germinal vesicle (GV) stage were collected from ovaries and cultured in modified NCSU37 up to 48 h. After culture, cumulus cells were removed and oocytes were parthenogenetically activated by 25 µm Ca2+ ionophore for 3 min and 2 mm 6-DMAP for 6 h. After activation, oocytes were further cultured up to the 2-cell stage. Groups of 30 oocytes were collected at each culture period for detection of IP3R1. According to our previous report in the mouse, IP3R1s were detected by western blotting using MPM-2 and Rbt03 antibody for detecting IP3R1 phosphorylation and total IP3R1 expression, respectively (Lee et al. 2006). In pig oocytes, IP3R1 was abundantly expressed at the GV stage. The total level of IP3R1 expression did not change during in vitro maturation or after activation. However, phosphorylated IP3R1 levels increased by 24 h although they were undetectable at the start of culture. Phosphorylation of IP3R1 reached maximal levels at 36 h. After activation, phosphorylation levels decreased progressively until the pronuclear (PN) stage. Phosphorylation of IP3R1 was observed at mitosis I to some extent. From these results, we detected for the first time IP3R1 expression and phosphorylation in pig oocytes. Moreover, our data suggest that phosphorylation of IP3R1 is dependent on cell cycle at least during meiosis, especially M-phase, as already shown for mouse oocytes. In vitro kinase assays for p34cdc2 kinase and MAPK will be carried out to clarify the relationship between IP3R1 phosphorylation and M-phase kinase(s).
258 THE EFFECT OF A PLANT PROTEIN COMPONENT OF MEDIA USED FOR BULL SPERM SEXING ON SPERM MEMBRANE STATUS
Authors: Bochenek M. , Smorag Z.
Published: January 2008

The aim of the work was to examine the effect of modified TALP medium (TALP/Pp, Animal Pharma B.V., Hengelo, The Netherlands)—used in the sperm sexing procedure—on bull sperm membrane status. The TALP was modified by replacement of bovine serum albumin (BSA) with a mixture of several plant proteins and soya lecithin (Pp). The Pp component was prepared using a high pressure homogenization process. The TALP/Pp had the same pH and osmotic pressure as the original TALP medium (TALP/BSA). The work was divided into 2 parts: (1) Nine ejaculates collected from 2 bulls (Holstein and Polish Red) were used. Immediately after collection, each ejaculate was split into 2 parts and diluted (1:2) with TALP/BSA or TALP/Pp. The sperm membrane status was examined after 3 days of storage at 15�C. (2) Fifteen ejaculates collected from 5 bulls (Holstein, Polish Red, and Simmental) were used. Each ejaculate was split into 2 parts: the first part was diluted with TALP/BSA, stained, incubated, and sexed according to the XY Inc. bull semen sexing procedure; the second part was diluted, stained, incubated, and collected after sexing into TALP/Pp with no egg yolk addition. In both groups no red food due was used to identify and exclude the dead spermatozoa from the sorted fractions. The sperm sexing procedure was performed with an SX MoFlo high-speed sorter at a speed of 3000–4000 cells/s. After collecting about 10 million spermatozoa, both fractions, X andY, were mixed, centrifuged at 700g for 15 min to concentrate the spermatozoa (20 million mL–1), and the sperm membranes examined. For sperm membrane examination, 'live/dead' samples were stained with SYBR-14/propidium iodide fluorochromes and analyzed by flow cytometry. The data from 20 000 spermatozoa were collected for each sample. The percentage of membrane-intact ('live') spermatozoa was taken for statistical analysis. The mean percentage of live spermatozoa stored for 3 days in TALP/BSA v. TALP/Pp was 25.7% (SD = 7.48) v. 28.58% (SD = 7.04), respectively (P < 0.01). The mean percentage of live spermatozoa in samples of sexed semen was 33.57% (SD = 18.97) for TALP/BSA and 38.51% (SD = 20.22) for TALP/Pp (P < 0.01). It can be concluded that Pp should be considered as a replacement for BSA in the TALP medium used for bull sperm sexing because (1) it results in significantly higher numbers of live spermatozoa after storage and/or sexing; (2) it eliminates a possible source of transmissible diseases (such as bovine spongiform encephalopathy); and (3) it decreases the total cost of the basic media used for the bull sperm sexing procedure.
266 BIRTH OF CANINE OFFSPRING FOLLOWING INSEMINATION OF A BITCH WITH FLOW-SORTED SPERMATOZOA
Authors: Meyers M. A. , Burns G. , Arn D. , Schenk J. L.
Published: January 2008

Different traits and temperaments that are sex-related often underlie the selection of guide dogs used for auditory and/or visually impaired humans. The objective of this project was to select X-chromosome-bearing canine sperm by means of flow cytometry/cell sorting based on DNA content (X chromosome- bearing canine sperm contain 3.7% more DNA than Y-chromosome-bearing sperm) and use the sorted sperm for artificial insemination (AI) to produce live offspring. This technology has proven reliable for sex selecting offspring using AI in a number of species and is of great commercial value to food animal livestock production. To test the efficacy of sorted canine sperm, a 2-year-old nulliparous Labrador retriever bitch in observed heat was monitored for plasma progesterone levels for a baseline rise to 2.8 ng mL–1, indicative of the LH surge. Progesterone levels more than doubled (5.8 ng mL–1) 2 days later, suggesting ovulation. One ejaculate was collected from a proven Labrador retriever stud on each of Days 2, 5, and 6 following the estimated LH peak. Sperm (200 million) were diluted in TRIS buffer without egg yolk (pH 7.4) and stained with 97.4 µm Hoechst 33342 in 1-mL aliquots for 45 min in a 34.5�C water bath and then diluted to 100 million sperm mL–1 with 4% egg yolk TRIS (pH 6.2) containing 0.002% food coloring dye (FD&C #40). Sperm were sex-sorted using an SX MoFlo sperm sorter (Dako, Fort Collins, CO, USA), operated at 40 psi and using a TRIS-based medium (pH = 6.8, 370 mOsm kg–1 water) as sheath fluid: 10 000–20 000 events s–1 and sort rates of 800–2000 sperm s–1. Sorted sperm were concentrated by centrifugation for 20 min at 850g and, after the supernatant was removed, were extended with 20% egg yolk TRIS extender (pH 6.8) to a total volume of 2 mL. Transcervical insemination of fresh, sorted sperm occurred within 9 h of the semen collection. An additional 1 mL of 20% egg yolk TRIS extender was used to flush the insemination catheter. The respective number of X-chromosome-bearing sperm inseminated per day was 46.2, 20.3, and 18.2 million, totaling 84.7 million sperm, and the weighted purity mean across the 3 sort days was 82%. Pregnancy was ascertained by ultrasound 30 days post-AI. Five puppies (3 female and 2 male) were delivered via natural birth 62 days after first AI. Only 60% of the offspring were of the predicted sex due to an 18% probability of chance, given the weighted mean purity of the sorted sperm and for a litter of 5 puppies. This demonstrates that fresh canine sperm can be separated by sex on the basis of DNA content, and transcervical insemination of low numbers of non-frozen sorted canine sperm can be used to produce live offspring.
This work was funded by XY, Inc.
273 REMOVAL OF ACROSOME FROM SPERM HEADS IMPROVES DEVELOPMENT OF RAT ZYGOTES THROUGH INTRACYTOPLASMIC SPERM INJECTION
Authors: Seita Y. , Sugio S. , Sano D. , Nakada M. , Hoshina M. , Okuda Y. , Ito J. , Kashiwazaki N.
Published: January 2008

In intracytoplasmic sperm injection (ICSI), sperm chromatin is introduced into the oocyte together with acrosome, which does not enter cytoplasm of oocytes during normal fertilization. In mice, acrosome of sperm head has a detrimental effect on embryonic development of the ICSI oocytes (Morozumi et al. 2006 Proc. Natl. Acad. Sci. USA 103, 17 661–17 666). We examined the effect of acrosome removal of frozen/thawed (F/T) rat sperm on development to term of the ICSI oocytes in order to improve production efficiency of live offspring from F/T rat spermatozoa through ICSI. In experiment 1, epididymal spermatozoa of the Wistar rats were frozen as described previously (Seita et al. 2005 Reprod. Fertil. Dev. 18, 256 abst). The F/T spermatozoa were sonicated to separate sperm heads. The sperm heads were divided into 3 treatment groups; nontreated sperm head (control), sperm heads exposed in 0.02% of Triton X-100 solution for 1 min (TX), sperm heads exposed in 0.02% lysolectin solution for 1 min (LL). Acrosomal status of the sperm heads was then examined by FITC-peanut agglutinin stain. In experiment 2, sperm heads of the control, TX, and LL treatments were microinjected into denuded oocytes obtained from superovulated females (Hirabayashi et al. 2002 Transgenic Res. 11, 221–228). The ICSI oocytes were cultured and observed for the formation of pronuclei (2PN) for 6 h and blastocyst formation at 120 h of culture. In experiment 3, the ICSI oocytes cultured for 6 h were transferred to recipient females to examine development to term. Statistical analyses of the results were carried out by 1-way ANOVA. In experiment 1, the TX (76%) and LL (89%) treatments showed higher rates of acrosome removal than that of the control (24%) group (P < 0.05). In experiment 2, the percentage of 2PN formation at 6 h after ICSI was not significantly different among sperm-treated groups, although the TX (76%) and LL (70%) groups were higher than the control (24%) group at 4 h (P < 0.05). The percentages of blastocyst formation were not significantly different among sperm-treated groups (control: 10%; TX: 25%; and LL: 25%). In experiment 3, the efficiency of development to term of the TX treatment group (21%: 12/57) was significantly higher than the control (5%: 3/55; P < 0.05), although the LL treatment group (16%: 12/75) was not significantly different from the control and TX treatment groups. These results indicate that acrosome removal using TX of F/T rat sperm heads before ICSI improves development to term of the ICSI oocytes. This benefical effect of acrosome removal may be due to the quick release of sperm-activating factors from sperm heads after ICSI in the rat.