bluuebunny/crossref_metadata_2025 huggingface dataset:
138 TIME-LAPSE CINEMATOGRAPHY-COMPATIBLE INJECTION-MOLDED MICROWELL CULTURE SYSTEM FOR TRACKING THE DEVELOPMENT OF INDIVIDUAL BOVINE EMBRYOS
Authors: Sugimura S. , Akai T. , Somfai T. , Hirayama M. , Aikawa Y. , Ohtake M. , Hattori H. , Kobayashi S. , Hashiyada Y. , Konishi K. , Imai K.
Published: August 2011

We have developed a polystyrene-based well of-the-well system (WOW) using injection moulding to track individual embryos throughout culture using time-lapse cinematography (TLC). The WOW-cultured bovine embryos following in vitro fertilization (IVF) were compared with conventional droplet (control)-cultured embryos on in vitro and in vivo development. Twenty-five of zygotes were cultured in each culture system containing 125 μL of CR1aa medium supplemented with 5% calf serum for 168 h after IVF. No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality, inner cell mass (ICM), and trophectoderm (TE) cell numbers and post-vitrification survival rates were not different between control- and WOW-derived blastocysts; however, incidence of apoptosis in the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at days 30 and 60 after embryo transfer (P < 0.05). The TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pick-up; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of 2 blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

This work was supported by the Research and Developmental Program for New Bio-Industry Initiatives.
142 CHIMERISM BY AGGREGATION OF BISECTED IN VITRO PRODUCED BOVINE EMBRYOS
Authors: Razza E. M. , Emanuelli I. P. , Barros C. M. , Nogueira M. F. G.
Published: August 2011

Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can be performed with whole or demi-embryos, in different stages of development and produced by in vivo or in vitro systems. However, aggregation efficiency tends to be reduced when using embryos in advanced stages (e.g. morulae and blastocysts). The aim of this work was to evaluate the effect of the agglutinating agent phytohemagglutinin-L (PHA) in the percentage of chimeras produced with in vitro-produced (IVP) bovine embryos. Cumulus–oocyte complexes (COC; 445; quality I and II) were matured in drops of 90 μL of TCM-199 bicarbonate supplemented with 10% of FCS and incubated for 22 to 24 h. Fertilization was performed in TALP-IVF medium for 18 h. Presumptive zygotes were transferred to SOF medium for in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. To conduct the manual bisection, embryos were placed into 3-μL microdrops of protein-free HEPES-buffered SOF medium. The bisection was executed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under stereomicroscope (35× magnification). Half-structures were joined and transferred to an embryo reconstruction plate, where they were kept for 3 min in drops containing 500 μg mL–1 phytohemagglutinin-L, before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells to in vitro culture. The structures were randomly allocated and the aggregation was performed between 2 whole (zona free) 8- to 16-cell stage embryos to construct aggregated chimeras in the presence [group (G)1, n = 32] or absence of PHA (G2, n = 34) and between demi-morula and demi-blastocyst with PHA (G3, n = 28) or without (G4, n = 29). The aggregation of structures was evaluated after 24 h. Aggregation rates among the 4 experimental groups and the main effects were analysed by Chi-square or Fisher’s exact test and significance was considered when P < 0.05. Embryo aggregation was higher in group G1 than G2 (75.0 and 50.0%, respectively; P = 0.045). Aggregation rate of demi-embryos was similar either in the presence (G3, 39.3%) or in the absence of PHA (G4, 20.7%; P = 0.16). The presence of PHA significantly increased the aggregation rates of the whole pre-compaction embryos (G1) compared with G3 (75.0 and 39.3%, respectively; P < 0.01). The use of PHA resulted in higher aggregation rates (58.3%) than non-use (36.5%; P = 0.03), whereas the embryonic stage of pre-compaction development (G1+G2) produced a higher rate of aggregation (62.1%) than post-compaction demi-embryos (G3+G4, 29.8%; P < 0.001). We could infer a positive effect of PHA on the aggregation rate of bovine IVP embryos only to the 8- to 16-cell stage of development.

Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).
147 USE OF EQUINE CHORIONIC GONADOTROPIN AFTER EMBRYO TRANSFER IN NELORE AND CROSSBRED RECIPIENT CATTLE
Authors: Mattos M. C. C. , Bastos M. R. , Oliveira A. C. S. , Gonçalves J. R. S. , Oliveira T. A. , Mourão G. B. , Sartori R.
Published: August 2011

Recent studies that have used eCG administration either 14 or 22 days after fixed-time AI in dairy and beef cattle have indicated a positive effect on conception rates. The aim of this study was to evaluate conception rates in embryo recipients treated with eCG 7 days after fixed-time embryo transfer (ET). A protocol for synchronization of ovulation was used in 470 Nelore and crossbred embryo recipient heifers (n = 179), dry cows (n = 152), and lactating cows (n = 139; body condition score = 3.0 to 3.5; >40 days in milk). All heifers and dry cows, but only 27.3% of lactating cows, were cycling at the beginning of the synchronization protocol. On Day 0, each female received IM injections of 2 mg of oestradiol benzoate (Gonadiol, Intervet Schering-Plough Animal Health, São Paulo, Brazil) and 0.075 mg of PGF2α (Preloban, Intervet Schering-Plough Animal Health). An intravaginal progesterone-releasing device (DIB, Intervet Schering-Plough Animal Health) was also inserted on Day 0. On Day 8, the DIB device was removed and cows or heifers received another IM treatment of 0.075 mg of PGF2α (Preloban), 300 IU of eCG (Novormon, Syntex S.A., Buenos Aires, Argentina) and 0.6 mg of oestradiol cipionate (Pfizer, Brazil). Transrectal ultrasonography was performed in all recipients on the day of ET (Day 16, 17, or 18 of the protocol) and only those with a corpus luteum greater than 15 mm in diameter were selected (n = 113 heifers, 114 dry cows, and 70 lactating cows). Transfers of fresh, frozen, and vitrified in vivo-produced embryos from Nelore donors were balanced between experimental groups. Seven days after ET, 127 animals received an IM injection of 400 IU of eCG (eCG group), and the 170 remaining cows or heifers were not treated with eCG, and were therefore considered the control group. Pregnancy diagnosis was performed 30 and 60 days after ovulation by transrectal ultrasound. Data were analysed using generalized linear models and results are presented as least squares means ± standard error. Conception rates were similar in the eCG and control groups at Day 30 (35.9 ± 5.5 and 33.5 ± 4.4, respectively; P > 0.10) and at Day 60 (27.6 ± 4.9 and 26.7 ± 3.9, respectively; P > 0.10). Contrary to other results in the literature, especially in dairy cows bred by fixed-time AI, the use of eCG 7 days after ET did not improve conception rates in Bos indicus cattle used as embryo recipients.

Financial support from CNPq, FAPESP, and Intervet Schering-Plough Animal Health of Brazil and Syntex S.A. of Argentina.
150 CONCEPTION RATES OF FRESH AND FROZEN IN VIVO-PRODUCED MORULAE AND BLASTOCYSTS IN LACTATING DAIRY RECIPIENT COWS
Authors: Sartori R. , Mattos M. C. C. , Bastos M. R. , Oliveira T. A. , Guenther J. N. , Wiltbank M. C.
Published: August 2011

The aim of this study was to evaluate conception rates of fresh or frozen embryos in the morula and blastocyst stages of development when transferred to lactating Holstein cows. During a 365-day period, 213 Day-7 embryos were produced by superovulation in lactating Holstein cows or nulliparous Holstein heifers and transferred to lactating Holstein cows synchronized with the Ovsynch protocol at the University of Wisconsin–Madison. After collection, embryos were evaluated and classified according to IETS standards. Subsequently, grades 1 and 2 embryos were conventionally frozen in ethylene glycol or transferred fresh to recipients on Day 7 of the cycle in the uterine horn ipsilateral to the corpus luteum. When there were more recipients than fresh embryos available, frozen embryos were thawed and transferred. Pregnancy diagnosis was performed on Days 30, 60, and 120 by ultrasound evaluations. Moreover, calving data were recorded. Data were analysed using generalized linear models and results are presented as least squares means ± standard errors. The average conception rates between 30 and 280 days of fresh (n = 45) and frozen (n = 168) embryos were similar (38.7 ± 7.3 and 29.4 ± 3.3%, respectively; P > 0.10). There was an interaction between treatment and embryo developmental stage (P < 0.10). The average conception rate was higher in fresh blastocysts (54.2 ± 12.8%) than fresh morulae (25.2 ± 5.1%) and cryopreserved embryos (P < 0.05). Moreover, there was no difference between conception rates of cryopreserved morulae and blastocysts (26.8 ± 3.6 and 32.1 ± 4.8%, respectively; P > 0.10). Embryo quality seems not to have influenced the conception rates of Holstein embryos (34.9 ± 3.3 and 32.9 ± 6.9% of grades 1 and 2, respectively; P > 0.10). We concluded, based on the results of this study, that cryopreservation of in vivo-produced embryos in Holstein donors did not substantially affect embryo viability and that the developmental stage of frozen embryos did not influence conception rates in lactating dairy recipients.

Financial support from CNPq and FAPESP of Brazil.
154 EVALUATION OF THE EFFECTIVENESS OF TREATMENT WITH TRYPSIN IN MURINES EMBRYOS EXPERIMENTALLY EXPOSED TO BOVINE HERPES VIRUS TYPE-1 (BoHV-1) BY THE NESTED-PCR TECHNIQUE
Authors: Palazzi E. , Pavão D. , Alves M. , Batista L. , Queiroz R. , Souza F. , D'Angelo M.
Published: August 2011

The cryopreservation of bovine embryos has allowed an increase in their commercialization causing concern about the transmission of pathogens through the production and transfer of in vitro embryos. The International Society of Embryo Transfer (IETS) suggests the treatment of embryos with trypsin or antibiotics in alternating washes with culture medium in order to remove and/or inactivate infectious agents that may interfere with the final product. The aim of this study was to evaluate the effectiveness of trypsin treatment in the elimination and/or removal of bovine herpes virus type-1 (BoHV-1), Colorado strain, in murine embryos. Viral detection was made by n-PCR and cytopathic effect in Madin Darby bovine kidney (MDBK) cells. Six- to 8-wk-old female mice (Swiss) were superovulated and mated with fertile males of the same strain. After 24 h, the zygotes (n = 262) were divided into 3 groups: control group submitted to sequential wash (CSW), the group exposed to the virus (30 μL; 106.5 virus mL–1) and submitted to sequential wash (ESW), and the group exposed to the virus and submitted to the trypsin treatment (ETT). All the groups of zygotes and the last sequential wash drops were tested by nested-PCR and inoculated in MDBK cells to allow observation of cytopathic effects. All groups except for CSW showed positive results for the nested-PCR for both zygotes and for the last wash drops. A cytopathic effect was observed in all groups except for CSW, demonstrating the viability of the virus after treatment. These results demonstrated that trypsin treatment was not effective in eliminating or removal of BoHV-1. The need for such studies is fundamentally important when considering the potential risk of pathogen transmission by reproductive biotechnologies.
158 DEVELOPMENT OF FIELD ASSAY FOR EVALUATION OF WHITE RHINOCEROS NEUTROPHIL FUNCTION AS A STRESS MARKER
Authors: Kruger M. , Pitts N. I. , Virgo J. , Betts E. , Delk K. , Fayrer-Hosken R. A.
Published: August 2011

Kruger National Park (KNP) is the primary source of translocated white rhinoceroses (Ceratotherium simum) in South Africa. Capture and transport of the rhinoceroses is a highly successful procedure. However, some relocated rhinoceroses present with fertility problems in the 1–2 years post capture. Novel research has shown that one can assess stress using respiratory neutrophil burst levels in several species (Huber et al. 2006 Protoplasma 229, 221–224; Weyts et al. 1998 Dev. Comp. Immunol. 22, 563–572). The hypothesis is that some rhinoceroses respond poorly to stress. The response to stress might be measurable by a neutrophil function (NF) assay, and NF depression might identify adversely stressed animals. The aim was to develop an in-the-bush test for NF and evaluate the changes in neutrophil activity during capture and loading into transportation crates. Neutrophil function was assayed using a portable luminometer (3M Clean-Trace™, 3M, St. Paul, MN, USA) while driving from one capture to the next. For the reaction mixture, 500 μL of PBS was incubated with 10 μL of fresh heparinized blood at 37.6°C. The incubator unit was power by a 230 V convertor in the vehicle. After 5 min of incubation, 100 μL of luminol was added. This sample was read in the luminometer as a blank and then to the sample, 100 μL of 12-O-tetradecanoylphorbol-13-acetate (TPA), also commonly known as phorbol 12-myristate 13-acetate (PMA), a diester of phorbol, was added. The luminescence readings or relative light units (RLU) were read at 2.5 min and then 5 min after PMA addition. All samples were prepared in triplicate. The readings were then taken every 5 min for 65 min. Two samples of blood were evaluated for each rhinoceros: the first sample at capture (anaesthetic induction) and then a second sample after loading of the rhinoceros into the transport crate, 20 to 30 min later. Two response curves were produced for each rhinoceros using the means of the triplicate readings. The curves were then transformed with a trend line and the area under the curve (AUC) was calculated. The 2 AUC for rhinoceros were then compared statistically using Wilcoxon rank regression, with P < 0.05 considered statistically different. Three distinct response curve patters were seen. Sixty-two percent (16/26) of the rhinoceroses had no statistical (P < 0.05) difference between capture and loading samples. In 5 of 26 (19%), the loading AUC was statistically greater than the capture sample, and in 5 of 26 (19%), the capture AUC was statistically greater than the loading sample. In conclusion, NF can be assayed in the field using the blood of wild caught rhinoceroses. The assay is repeatable and can distinguish 3 populations of wild caught rhinoceroses. The hope is that future research will allow us to identify rhinoceroses that have negative stress reactions and to change the capture conditions to make the process less stressful.
159 COMPARISON BETWEEN EQUINE CHORIONIC GONADOTROPIN AND PORCINE FOLLICLE STIMULATING HORMONE FOR IN VIVO PRODUCTION OF EMBRYOS IN ALPACAS (VICUGNA PACOS) SHOWING NATURAL LUTEAL PHASE AFTER INDUCTION OF OVULATION
Authors: Vivanco W. , Huaman E. , Leon S. , Nunez T. , Gregoire A. , Ponce D. , Alvarado E. , Asparrin M.
Published: August 2011

Alpacas are animals with induced ovulation, andthey show high individual variation in the symptoms, duration, and regularity of oestrus or period of female receptivity to males; their follicular phase does not end in ovulation and subsequent luteal phase unless an external stimulation such as copulation or exogenous application of an ovulation inducing hormone is applied. The objective of the present study was to compare the use of eCG v. porcine (p)FSH as superovulatory hormones for the in vivo production of embryos in alpacas that were selected as being receptive to the male and were treated with an ovulation-inducing hormone to generate a luteal phase. Twenty adult (3 to 5 years old) female alpacas, located at Mallkini, Puno, Peru (at 4100 m elevation), were used for the trial. A group of females was exposed to males to test for breeding receptivity; 20 alpacas were receptive, adopting copulatory position. Each of the selected females received 3.75 mg of LH IM (Lutropin®, Bioniche Animal Health, Belleville, ON, Canada). Day 0 was then considered the date of LH injection. The 20 alpacas were then distributed into 2 treatments: Treatment 1 (T1 = 10 alpacas) received on Day 2, 1000 IU of eCG IM (Pregnecol®, Bioniche Animal Health) and on Day 7, a dose of PGF2α IM (0.263 mg of cloprostenol; Ciclar®, Andeanvet-Zoovet, Lima, Peru). Treatment 2 alpacas (T2 = 10 alpacas) received from Day 2 and up to Day 5, at 12-h interval, decreasing doses of pFSH IM (100 mg; Folltropin V®, Bioniche Animal Health) for 4 days, and on Day 7, a dose of PGF2α IM (0.263 mg of cloprostenol; Ciclar®, Andeanvet-Zoovet). All alpacas from T1 and T2 were mated twice with fertile males, the first mating at 24 h after the injection of PGF2α and the second at 12 h after the first mating. All females received a dose of GnRH IM (0.0084 mg of buserelin; Buserelina®, Andeanvet-Zoovet) at time of first mating. The embryos in both treatments were collected 6.5 days after the first mating by nonsurgical transcervical embryo flushing. There were no significant differences in the mean number of blastocysts collected per treatment (P > 0.05), being 3.0 ± 2.87 blastocyst for T1 and 1.6 ± 2.67 for T2. The number of blastocysts per treatment was 30 and 16 for T1 and T2, respectively. The results show that superovulatory treatment with eCG is more effective for the production of viable blastocysts than treatment with pFSH in alpacas treated for superovulation during the luteal phase.

This work was partially funded by Bioniche Animal Health.
16 FERTILITY RATE OBTAINED WITH A NEW INTRAVAGINAL PROGESTERONE RELEASING DEVICE DICO IN SHEEP
Authors: García-Pintos C. , dos Santos-Neto P. C. , Menchaca A.
Published: August 2011

A new intravaginal device DICO (Dispositivo Intravaginal Caprino Ovino, 0.3 g progesterone; Syntex, Buenos Aires, Argentina) consisting of a progesterone impregnated medical silicone was developed for oestrus synchronization in sheep and goats. The effectiveness of this device to control serum progesterone concentrations, follicular development and time of ovulation has been recently reported in ewes (Vilariño et al. 2010 Small Rumin. Res. 91, 219–224); however, fertility obtained with DICO has not been evaluated yet. The aim of this study was to determine fertility rate obtained with DICO by fixed-time cervical or intrauterine insemination in sheep. The experiment was performed on a total of 569 Corriedale multiparous ewes in breeding season, with a body condition score of 3.0 ± 0.1 (scale 0–5). Ewes received a Short-term Protocol consisting of a 6 days of treatment with DICO (n = 174), CIDR-G (0.3 g of progesterone, Pfizer, NY, USA; n = 197) or sponges with 60 mg of medroxiprogesterone acetate (MAP, Syntex; n = 198). One dose of prostaglandin F2α analogue (125 μg of cloprostenol, Cilcase DL, Syntex) and 300 IU of eCG (Novormon, Syntex) was given at the time of device withdrawal. Insemination was performed 48 or 54 h after device removal by cervical (200 millions of spermatozoa) or laparoscopic intrauterine insemination (100 millions of spermatozoa), respectively. Pregnancy diagnosis was performed by transrectal ultrasonography (5 MHz, Well D, Beijing, China) 40 days after insemination. Pregnancy rate (pregnant ewes/inseminated ewes) was compared by logistic regression, and number of fetuses/pregnant ewes was analysed by Poisson regression. Regarding pregnancy rate, a significant difference (P < 0.05) was found between cervical (39.5%, 109/276) v. intrauterine insemination (58.3%, 172/295), as well as between progesterone devices (DICO: 55.7%, 97/174; CIDR-G: 55.8%, 110/197) v. MAP sponges (37.4%, 74/198). The results are shown in Table 1. In conclusion, the current study shows that the new intravaginal device DICO is useful to obtain acceptable fertility with fixed-time insemination in sheep. When cervical insemination at 48 h or intrauterine insemination at 54 h after device removal is performed, similar or better results than CIDR-G or MAP sponges are achieved, respectively.

Table 1.Results obtained with DICO, CIDR and sponges after cervical or intrauterine insemination in sheep

Supported by Syntex.
160 SUCCESSFUL PREGNANCY IN ESTRUS-SYNCHRONIZED HIMALAYAN TAHRS (HEMITRAGUS JEMLAHICUS) BY TRANSCERVICALLY INSEMINATING FRESH AND EXTENDED-CHILLED SEMEN COLLECTED BY ELECTROEJACULATION
Authors: Yong H. , Bae B.-S. , Kim S.-D. , Hyun S.-H.
Published: August 2011

In December 2009, 8 female Himalayan tahrs (Hemitragus jemlahicus) were synchronized by inserting CIDR devices for 13 or 15 days. One day before removal of the CIDRs, a combination of pregnant mare serum gonadotropin (PMSG) 400 IU and hCG 200 IU (PG600®, Intervet, Boxmeer, the Netherlands) was intramuscularly injected into each animal and PGF2α was injected at the time of CIDR withdrawal. Semen was collected by electrical stimulation, diluted, cooled to 5°C for 2 h, and maintained in a refrigerator before performing AI twice. Pregnancy diagnosis was conducted by Doppler ultrasonography on March 2, 2010. Four female Himalayan tahrs were impregnated, showing a fetus present in their uteri. In the first experiment in which CIDRs were inserted into the vagina of 4 Himalayan tahrs for 13 days, 3 of the tahrs were transcervically inseminated with fresh semen, whereas 1 was inseminated with frozen–thawed semen 33 h after CIDR removal. As a second insemination experiment, 46 h after CIDR removal, all 4 tahrs were inseminated with diluted (1:3) fresh semen. While the tahr that was later inseminated with frozen–thawed semen did not become pregnant, the other 3 Himalayan tahrs became pregnant. In the second experiment, CIDR devices were inserted into the vaginas of 4 Himalayan tahrs for 15 days. Extended (1:7)-chilled semen was used for transcervical insemination 42 and 49 h after CIDR withdrawal. When performing the second insemination, it was difficult or impossible to penetrate cervical canals. Thus, only 1 tahr became pregnant. These findings show 13 days of CIDR insertion, the dose of PMSG 400 IU and hCG 200 IU, and the use of PGF2α as beneficial for inducing oestrus synchronization, and prove that extended-chilled semen may also be used for artificial breeding of Himalayan tahrs. This is the first report of successful pregnancy induced by artificial insemination of fresh or extended-chilled semen in oestrus-synchronized Himalayan tahrs.
163 IN VITRO CULTURE OF PORCINE OOCYTE-GRANULOSA CELL COMPLEXES
Authors: Diao Y. F. , Han R. X. , Kim H. R. , Park C. S. , Jin D. I.
Published: August 2011

The objective of this study was to investigate the effects of porcine follicular fluid (PFF) and insulin-like growth factor-1 (IGF-1) on the growth of porcine oocyte-granulosa cell complexes (OGC) in vitro, in an effort to improve meiotic and developmental competence in vitro. Porcine OGC were manually dissected from early antral follicles of diameter 400 to 700 μm, and intact oocytes with undamaged granulosa cells were selected for culture. Between 3 and 5 OGC were combined in 50-uL droplets and cultured for 12 days in M199 medium supplemented with PVP, oestradiol, FSH, transferrin, L-ascorbic acid, and insulin. The OGC were cultured at 38.5°C in a humidified atmosphere of 5% CO2 in air for 12 days, and the oocytes were matured for 44 h. Oocyte diameter, exclusive of the zona pellucida, was measured on day 0 and day 12 of culture. Control group was cultured in the absence of PFF or IGF-1. The experiment was divided into 2 parts. In part 1, treatment groups were cultured with 2.5, 5.0, or 7.5% (all v/v) PFF. Control OGC grew as spheres that were formed by granulosa cells. In treatment groups, the granulosa cells spread and grew on the bottom of dishes. When oocyte diameter was measured after 12 days of culture, no significant difference among groups was observed (104.07, 103.96, and 104.27 μm at the 3 PFF concentrations used; control: 104.03 μm). Similarly, the survival rate of oocytes did not differ significantly among groups. However, survival rate fell somewhat in the group treated with PFF (control: 65%; tests: 58.87, 58.33, and 50.83% at the 3 PFF concentrations used). The maturation rates of oocytes in the control was significantly higher than those of the treatment groups [25.83% (control) v. 14, 11.67, and 3.67% (the 3 treatment groups); P < 0.05]. Thus, the first conclusion is that supplementation of culture medium with PFF did not enhance the development of porcine OGC in vitro. In part 2 of the experiment, treatment groups were cultured with 10, 50, or 100 ng mL–1 IGF-1. The percentages of OGC showing antrum formation were 80, 80, 100, and 100% in groups treated with 0, 10, 50, or 100 ng mL–1 IGF-1, respectively. Average oocyte diameter was 94.16 to 94.58 μm just after OGC collection. However, the average diameter of oocytes cultured for 12 days with 50 or 100 ng mL–1 IGF-1 was significantly higher than that of the control or 10 ng mL–1 groups [108.88 and 108.31 μm (50 and 100 ng mL–1 IGF-1) v. 105.98 μm and 106.67 μm (0 and 10 ng mL–1 IGF-1); P < 0.05]. The maturation rate of oocytes grown with 10 and 50 ng mL–1 IGF-1 was higher than those of the other 2 groups [30 and 40.6% (10 and 50 ng mL–1 IGF-1, respectively) v. 25 and 26% (0 and 100 ng mL–1 IGF-1, respectively); P < 0.05]. Thus, the second conclusion is that 50 ng mL–1 IGF-1 improves the growth and maturation of porcine oocyte-granulosa cell complexes in vitro.
164 EPIGENETIC ANALYSIS OF GENOMIC DNA IN PREPUBERAL AND ADULT BOVINE OOCYTES
Authors: Diederich M. , Heinzmann J. , Kues W. , Baulain U. , Haaf T. , Reinhardt R. , Niemann H.
Published: August 2011

The use of oocytes obtained from prepuberal cattle shortens the generation interval by producing descendants of genetically valuable animals before achieving actual cultivation maturity. However, several studies proved that oocytes derived from prepuberal animals differ significantly from oocytes of adult animals with regard to their developmental capability and therefore reproductive potential. Epigenetic events are taken into consideration as a possible reason for this phenomenon. Particularly DNA methylation, allele specific gene expression in a parent-of-origin-specific manner (imprinting), and certain histone modifications, like acetylations, carboxylations, and phosphorylations, play an important role. This project aims to gain knowledge about the mechanisms involved in attaining of the full developmental potential of bovine oocytes. Using immature and in vitro matured oocytes of prepuberal and adult cattle, a comparative study was conducted by measuring mRNA expression of 4 developmentally relevant, but non-imprinted genes (GDF9, GLUT1, PRDX1, and ZAR1) as well as the general DNA methylation status, performed by bisulfite sequencing of 2 satellite sequences [bovine testis satellite I DNA segment 2 (BTSS2) and Bos taurus α satellite I DNA (BTS)]. After various pretreatments, immature bovine oocytes were collected from prepuberal calves [6–9 months, either left untreated (Ca1) or treated with FSH (Ca2) or FSH+IGF1 (Ca3) or FSH+IGFK (Ca4)] and adult animals [≥2nd lactation, either left untreated (Ad1) or treated with FSH (Ad2)] using the Ovum-pick-up (OPU) technique. The Ad1 group was considered the control group. First results of the qPCR analyses of immature oocytes show differences between treatment groups for GLUT1, PRDX1, and ZAR1 transcripts. Compared with Ad1, GLUT1 expression increased in Ad2 [fold change (FC) 2.2], Ca1 (FC 2.0), Ca2 (FC 1.8), and Ca3 (FC 1.4). The genes PRDX1 and ZAR1 were reduced in all groups by 0.02 to 0.07 in comparison with Ad1. The GDF9 showed generally a very low expression. The methylation analysis shows for BTSS2 and BTS significant differences before and after in vitro maturation in the groups Ad1 (BTSS2: 49.6 v. 64.9%), Ad2 (BTS: 76.7 v. 52.5%), Ca1 (BTSS2: 74.6 v. 53.3%), Ca2 (BTS: 72.8 v. 57.8%) and Ca3 (BTSS2: 60.6 v. 71.7%). Currently, the first experiment and statistical analysis are under way. The preliminary data confirm differences in gene expression between prepuberal and adult animals, and demonstrates the dependence of the methylation pattern on age and maturation status. These results contribute to a better understanding of the developmental potential of prepuberal oocytes in order to optimize their use for in vitro production of embryos.

This work was supported by the H. Wilhelm Schaumann Foundation, Hamburg.
165 SHORT-TIME FASTING AFFECTS METABOLIC MARKERS WITHOUT IMPACT ON FOLLICLE AND OOCYTE DEVELOPMENT IN THE RABBIT MODEL
Authors: Garcia-Garcia R. M. , Rebollar P. G. , Arias Alvarez M. , Sakr O. G. , Ramos Ibeas P. , Brecchia G. , Millan P. , Boiti C. , Lorenzo P. L.
Published: August 2011

Our goal was to elucidate whether acute energy restriction has any influence on ovarian reproductive processes and the relationship with some metabolic markers in the rabbit model. A total of 22 nulliparous rabbits were randomly distributed in 2 groups: control group fed ad libitum (C; n = 12) and 72-h fasted group (F; n = 10). Before and after the deprivation period, body weight (BW), estimated body composition, serum triglycerides (TG), nonesterified fatty acids (NEFA), and leptin concentrations were measured. After fasting, ovaries were retrieved: one was used to study oocyte in vitro nuclear and cytoplasmic maturation (in terms of granule cortical migration; Arias-Alvarez et al. 2010 Theriogenology 73, 26–35) and the other one was processed histologically to assess follicle categorization and atresia rate by TUNEL and by immunolocalization of procaspase-3 (PC3). Statistical analysis was carried out by MIXED and CATMOD procedures of SAS program. The BW of control rabbits increased at the end of the experiment (3.96 ± 0.06 v. 4.03 ± 0.09 kg; P < 0.05), but the fasted group showed similar weight compared with 72 h before and the C group (3.97 ± 0.09 kg). No differences were observed on estimated body composition between C and F animals (61.0 ± 0.52% water, 2.96 ± 0.02% ash, 18.6 ± 0.51% lipids, 18.4 ± 0.05% protein, and 1206 ± 21.6 kJ/100 g of energy). The TG was similar between groups (74.4 ± 9.9 v. 49.3 ± 10.4 mg dL–1), whereas NEFA concentrations were significantly higher in the F group than before fasting (0.29 ± 0.04 mmol L–1 v. 0.17 ± 0.03 mmol L–1; P < 0.05) and than in the control group (0.09 ± 0.04 mmol L–1; P < 0.05). In opposition, leptin concentrations were lower in the F group than before fasting (2.2 ± 0.6 v. 5.1 ± 0.4; P < 0.05) and than in C rabbits after fasting (4.6 ± 0.5 ng mL–1; P < 0.05). No differences were found in the number of primordial (192.0 ± 52.9 v. 276.0 ± 88.6), primary (36.7 ± 6.6 v. 45.5 ± 7.8), secondary (37.1 ± 7.5 v. 54.1 ± 14.6), and antral follicles (21.9 ± 2.1 v. 22.3 ± 2.2) between the C and F group. The rates of healthy (53.2 ± 4.3 v. 57.4 ± 5.0), early atretic measured as <50% of apoptotic cells (15.4 ± 2.2 v. 17.2 ± 2.6), and late follicles measured as >50% of apoptotic cells (31.4 ± 3.6 v. 25.4 ± 4.2) did not differ between groups. Cytoplasm staining of PC3 was observed in stroma, follicles of all sizes, and corpora lutea of both groups. Atretic follicles did not exhibit immunoreactivity to PC3. No significant differences were observed in nuclear maturation and migrated cortical granule rates (63.6 v. 53.7% and 12.0 v. 4.7%, respectively). In conclusion, acute fasting affects BW and some metabolic markers (NEFA and leptin), but follicle and oocyte development is not impaired in our rabbit model.

We acknowledge CM, FSE, and MEC for funding. This work is part of an integrated Action between Spain and Italy.
169 CHANGES OF PROTEIN PROFILES DURING FOLLICLE DEVELOPMENT AND IN VITRO OOCYTE MATURATION IN THE PIG
Authors: Ji M. R. , Jang D. M. , Lee Y. S. , Cheong H. T. , Yang B. K. , Park C. K.
Published: August 2011

When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro conditions is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development. In the first study, to investigate protein patterns during oocyte maturation in vitro, immature oocytes (germinal vesicle stage; GV stage) as control and oocytes matured (M-II phase) in vitro were analysed. The porcine oocytes in the GV stage were put in culture with TCM-199 for 44 h for M-II-stage oocytes. Total proteins were extracted from 1200 oocytes for GV and M-II stages, separated on 2-D gels, and stained with silver. In general, the overall protein staining pattern between the 2 gels was remarkably similar for most protein spots. Analysis of the gels identified proteins that were up- or down-regulated between GV and M-II stages. Up-regulated proteins were identified as PDE4D, GPKOW, PGM5, HSP70, ZPG4, galK1, GST-β, PDX1, PDX2, and PDX3. In contrast, down-regulated proteins were identified as PRKAB1, GRP78, TD-pozl, ERP57, MPP1, DTNA, ZP3B, HSP90, HSP86, and HSP27. This study has identified a novel protein, named myomegalin, that interacts with cyclic nucleotide phosphodiesterase (PDE4D). The second study analysed changes in proteins in follicular fluids during porcine follicular development. Follicular fluids were collected from follicles of 1- to 2-, 2- to 6-, and 6- to 10-mm diameter from ovaries of slaughtered pigs. Total proteins were extracted from follicular fluids by M-PER Mammalian Protein Extraction Reagent. Differentially expressed proteins were analysed by MALDI mass spectrometry and searched on NCBInr. As a result, Spot No. 28 from the 2- to 6-mm follicle was Ig lambda chain C region, and Spot No. 32 and 33 from 6- to 10-mm were Apolipoprotein A-IV (APOA4). Increases of those proteins were correlated with follicular development. These results indicate that in vitro maturation changes the protein profile of porcine oocytes, which play important roles in the sequence of molecular events in porcine oocyte maturation and follicular development.

This work was supported by Korean Research Foundation Grant funded by the Korean Government (2009-0071610).
170 NATURALLY OCCURRING CHRONIC MASTITIS COMPROMISES FOLLICULOGENESIS, AFFECTS VASCULARIZATION, AND INTERACTS WITH DIFFERENTIATION FACTOR GDF-9 IN BOVINE OVARIAN STROMA
Authors: Rahman M. M. , Mazilli M. , Pennarossa G. , Brevini T. A. L. , Vanelli A. , Zecconi A. , Gandolfi F.
Published: August 2011

Recent studies have suggested an association between reproductive failure and mastitis in lactating dairy cows, but our understanding of how mastitis affects reproduction is still limited. In the present study we investigated the effects of naturally occurring chronic mastitis on the population dynamics of ovarian follicles. Ovaries and milk samples were collected from 74 cows at slaughter. Milk samples from each quarter, were analyzed following National Mastitis Council procedures. Based on the presence of major pathogens and somatic cell count results, animals were sorted in 9 groups, but only the 2 extremes were considered for further analysis: uninfected (n = 8) and affected by chronic mastitis (n = 9). Primordial, primary, and secondary follicles were counted and scored on similar surface area of ovary sections for each animal (mean ± SD = 5.65 ± 0.25 cm2). They were analyzed with Fisher’s exact test, and the association between health status and follicle number was estimated by odds ratios ± confidence limits. Vasculature area in the ovarian cortex of healthy and mastitic animals was identified using Bandeiraea simplicifolia-I lectin (BSL-I). Results were quantified with the dedicated software MacBiophotonics image J, NIH, USA, and subsequently analyzed with t-test for statistical significance. Follicles were further characterized by immunostaining with a GDF-9-specific antibody. The intensity of the staining was semi-quantified using a relative scale: 0, 1, and 2 for no, weak, and strong staining, respectively. Our results indicate no (P > 0.05) difference between the numbers of primordial and primary follicles in healthy and affected animals. In contrast, the number of secondary follicles was significantly lower in sick animals (odds ratio 10.50*; P < 0.05), indicating 10 times higher risk for a mastitic animal to have less than 2 secondary follicles per square centimeter. Ovarian stromal vasculature represented the 6.38 ± 0.66% of cortical area in healthy animals v. 4.24 ± 0.37% (P < 0.001) in affected cows. The GDF-9 immunostaining revealed decreased fluorescence intensity in mastitic animals. Our results show that chronic mastitis is associated with considerable alterations in follicle growth and differentiation with a decreased ability of primary follicles to develop into the secondary state in affected animals. This is accompanied by a significant decrease of ovarian vasculature and the down-regulation of the follicle differentiation-associated factor GDF. The present findings substantiate the hypothesis that mastitis can reduce fertility by exerting a negative effect on ovarian function.
174 EVALUATION OF THE 9-DAY PROTOCOL FOR ESTROUS SYNCHRONIZATION IN SANTA INÊS EWES
Authors: Viau P. , Paes de Barros M. B. , Dias L. M. K. , Nicolau S. S. , Marino C. T. , Oliveira C. A.
Published: August 2011

The objective of the present study was to characterise follicular dynamics in Santa Ines (SI) during a 9-day protocol for oestrous synchronization and to evaluate the efficiency of the vaginal progesterone-releasing device Primer-PR® (Tecnopec, Brazil). Cyclic females were used (n = 10) at the ruminant sector of the veterinary hospital of the FMVZ–USP, in March 2009. The animals had body scores between 2.5 and 3 and were of proven fertility. Ultrasound examinations were performed using an ALOKA SSD-500 Scanner (Berger, Brazil) with a linear 5.0-MHz transducer, attached to a handle to allow safe intrarectal manipulation. Examinations were performed daily from 3 days (D-3) before the day of device placement (D0) until the day of device removal (D9), and twice daily from device removal until ovulation. All ewes received 0.03 mg of prostaglandin (D-cloprostenol, Prolise®, Tecnopec, Brazil) on D9. Ovulation was assessed by the disappearance of the growing larger follicle or follicles present in the previous examinations. Oestrous detection was performed using an intact male 3 times a day (at 10:00 a.m., 6:00 p.m., and 12:00 p.m.) from D9 until the last acceptant ewe, and blood samples were taken by jugular puncture for progesterone (P4) measurement by radioimmunoassay on solid phase (COAT-A-COUNT, Siemens, USA) from D-3 until the day of ovulation. Data were analysed by Shapiro–Wilk (PROC UNIVARIATE) using the SAS program (SAS Institute Inc., Cary, NC, USA, 2001) and shown as mean ± standard deviation. Plasma P4 concentrations between D0 and D9 were 6.56 ± 2.32 ng mL–1, peaking between D3 and D5 (8.07 ± 2.31 ng mL–1). Oestrous behaviour was shown 45.6 ± 12.71 h after Primer-PR® removal. The first and the last ewe that showed oestrous behaviour was at 30 h and 66 h after Primer-PR® removal, respectively, and the majority of ewes (50%) at 42 h. Oestrous lasted 26.40 ± 9.47 h, and the majority of ewes (70%) showed oestrous behaviour during 24 h. Ovulation occurred 73 ± 14.38 h after Primer–PR® removal and 1.3 ± 0.48 ovulations per animal were observed. From all growing presumptively dominant follicles observed, 92.3% of them ovulated. When double ovulations occurred (n = 3), the interval between first and second ovulation was 16 ± 6.93 h. Emergence of the ovulatory follicular wave occurred at 8.5 days + 16 h after Primer–PR® insertion. The follicles observed to continue growing had a diameter of 3.48 ± 0.28 mm when they were first detected and reached 5.63 ± 0.66 mm, with a growth rate of 0.73 ± 0.43 mm per day. A standard follicle wave within the 9-day protocol was not possible to determine, and the follicles receded in up to 4 days. In conclusion, placement of a Primer-PR® device for 9 days resulted in synchronous oestrus and ovulation in Santa Inês ewes.

FAPESP, CAPES, Tecnopec.
180 HEAT STRESS INDUCES ALTERATION IN EXPRESSION OF GENES RELATED TO COMPETENCE AND IMPLANTATION IN NELORE BOVINE IN VITRO-PRODUCED EMBRYOS
Authors: Silva C. F. , Castilho A. C. S. , Satrapa R. A. , Puelker R. Z. , Razza E. M. , Eduardo H. P. , Buratini J. , Barros C. M.
Published: August 2011

Several factors affect early embryonic development in cattle, including heat stress. These factors can contribute to high early embryonic loss, probably altering gene expression. Studies using microarray-profiled genome-wide RNA expression for in vitro-produced blastocysts have compared embryos resulting in calf delivery or no pregnancy, and they have identified genes with potential roles in pregnancy and embryo competence. The aim of the present work was to compare the expression of some genes (PLAC8, HSF1, COX-2, and CDX-2) related to embryo competence and embryonic implantation between in vitro-produced embryos from the Nelore breed (Bos indicus), submitted or not submitted to heat stress. Oocytes from Nelore cows were aspirated by ovum pickup and matured for 22 h (TCM-199 with bicarbonate, supplemented with 10% FCS, 2 μL mL–1 of pyruvate, 75 μg mL–1 of amicacin, 20 μg mL–1 of FSH, and 2 IU mL–1 of hCG) at 38.5°C with 5% CO2 in air. The fertilization (Day 0) was performed with semen from Nellore bulls. After a 12-h fertilization period, in Tyrode’s lactate stock medium supplemented with 6 mg mL–1 of BSA, 2 mL mL–1 of pyruvate, 75 mg mL–1 of amicacin, 11 mg mL–1 of heparin, and 44 mL mL–1 of phenylalanine solution, presumptive zygotes were denuded and randomly divided in 2 groups: nonstressed and stressed. The culture medium was SOFaaci supplemented with sodium pyruvate (0, 2%), 5 mg mL–1 of BSA and 5% FCS. Embryo culture was performed at 38.5°C, 90% N2, 5% CO2, and 5% O2. In the stressed group, 96 h after fertilization, the embryos were subjected to heat stress of 41°C for 6 consecutive hours and then returned to a temperature of 38.5°C. On Day 7, pools of 5 blastocysts (nonstressed, n = 9; stressed, n = 7) were submitted to total RNA extraction (RNeasy, Qiagen, Valencia, CA). The gene expression of target genes was measured by real-time RT-PCR with oligo-dT in the reverse transcription and bovine-specific primers in the PCR. Expression of cyclophlin A was used as an internal control. The means of mRNA levels of target genes between the groups were compared by t-test. The PLAC8 mRNA levels were higher in nonstressed blastocysts in comparison with the stressed group. The HSF1 and CDX2 mRNA was detectable only in nonstressed embryos. The COX2 mRNA levels did not differ between groups. The higher levels of PLAC8 and the CDX2 expression on nonstressed embryos indicate better competence of embryos not submitted to heat stress. Furthermore, the absence of HSF1 mRNA in the stressed embryos does not reflect the lack of biological activity of this protein. In conclusion, the data indicate that heat stress alters the gene expression pattern of in vitro-produced embryos in the Nelore breed.

FAPESP (São Paulo, Brazil) is acknowledged for funding and fellowships for Castilho, Satrapa, and Razza.
181 TRANSCRIPTIONAL SEXUAL DIMORPHISM IN AUTOSOMAL GENES ON BOVINE DAY 14 EMBRYOS
Authors: Bermejo-Alvarez P. , Rizos D. , Lonergan P. , Adan A.-G.
Published: August 2011

In bovine blastocysts, a large transcriptional sexual dimorphism affecting almost one-third of the genes is observed. At this early stage, sex chromosomes impose extensive transcriptional regulation on autosomal genes. However, little is known about the situation in further stages of embryo development. The aim of the present study was to determine the transcriptional sexual dimorphism on Day 14 bovine embryos for 10 candidate genes. In vitro-produced bovine blastocysts were transferred on Day 7 to the ipsilateral uterine horn of synchronized recipients. Seven days after embryo transfer, the recipients were slaughtered and Day 14 embryos were recovered by uterine flushing. Fourteen embryos of a similar size were selected for the analysis (8 males, 6 females). PolyA mRNA and cDNA synthesis and DNA extraction were performed individually. Embryo sexing was performed based on the expression of the Y-linked gene YZRSR2, and the result was confirmed on DNA by a PCR for BRY/Sat. The mRNA abundance relative to the housekeeping H2AZ was analysed for 5 genes known to display transcriptional sexual dimorphism at the blastocyst stage: the X-linked gene glucose-6-phosphate dehydrogenase (G6PD); the autosomal genes DNA methyltransferase 3 A (DNMT3A), gluthatione S-transferase mu 3 (GSTM3), laminin α 1 (LAMA1), progesterone receptor membrane component 1 (PGRMC1), and interferon tau (IFNT2); and 4 genes related to sex determination and differentiation: Wilms tumour 1 (WT1), GATA binding protein 4 (GATA4), zinc finger protein, multitype 2 (ZFPM2, also known as FOG2), and double-sex and mab-3-related transcription factor 1 (DMRT1). Statistical differences were assessed by ANOVA (P ≤ 0.05). Consistently with the situation observed in blastocysts, DNMT3A was significantly up-regulated in males (2.5 ± 0.5 v. 1 ± 0.2), and IFNT2 in females (1 ± 0.2 v. 1.8 ± 0.5), but G6PD, GSTM3, LAMA1, and PGRMC1 did not show significant differences. Among the genes linked with sex determination, WT1 was significantly up-regulated in males (1.7 ± 0.2 v. 1 ± 0.2), whereas the opposite situation was observed for GATA4 (1 ± 0.2 v. 1.5 ± 0.2). No differences between sexes were observed for ZFPM2 (1 ± 0.3 v. 1.1 ± 0.3) and DMRT1 (1.2 ± 0.2 v. 1 ± 0.1). In conclusion, transcriptional sexual dimorphism seems to be reduced in more advanced stages of embryo development, but 2 genes, related to DNA methylation (DNMT3A) and pregnancy recognition (IFNT2), maintained the sex-specific differences observed at the blastocyst stage. Furthermore, the expression of 3 potential activators of SRY (WT1, GATA4, and ZFPM2) and the regulator of male development DMRT1 was observed on Day 14 bovine conceptuses, and 2 of the genes (WT1 and GATA4) showed sex-specific transcriptional regulation.

This work was funded by Science Foundation Ireland, AGL2009-11358, AGL2009-11810 funding from the Spanish Ministry of Science and Technology.