bluuebunny/crossref_metadata_2025 huggingface dataset:
113 EVALUATION OF DIFFERENT CRYOPROTECTANT AND FORSKOLIN IN THE CULTURE MEDIUM FOR IMPROVING THE EFFICACY OF VITRIFICATION OF BOS INDICUS IN VITRO-DERIVED EMBRYOS
Authors: Sanches B. V. , Filho B. D. O. , Pontes J. H. F. , Basso A. C. , Meirinhos M. L. G. , Ferreira C. R. , Chiaratti M. R. , Viu M. A. O. , Seneda M. M.
Published: August 2010

Embryo cryopreservation is an essential method for the biotechnology of reproduction. This is the safest option for interchange of genetic material for research and commercial purposes. For cattle, Brazil has become the leading country in the world for the number of in vitro-produced embryos, using mostly Bos indicus animals. However, considering the in vitro method of embryo production, field results have shown a lower resistance to cryopreservation for B. indicus when compared with Bos taurus embryos. A possible explanation for this is a great concentration of lipid droplets in the cytoplasm of cells fromB. indicus embryos. The objective of this study was to compare 2 cryoprotectants (Propanediol and DMSO) to vitrification and evaluate the effect of adding 10 μM forskolin to the SOF medium for embryo culture before cryopreservation. For all the experiments, ovaries from slaughtered Nelore Bos indicus donors were recovered and maintained at 30 to 35°C in NaCl solution until recovery of the COC. Embryos submmited to vitrification were expanded blastocysts at Day 7 of in vitro culture. In the first experiment embryos were first incubated in 10% ethylene glycol (EG) plus 10% DMSO dissolved in holding medium (TCM-HEPES with 20% calf serum) for 1 min and then transferred to droplet of 20% EG plus 20% DMSO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen (n = 107; group EG + DMSO). For the group EG + Propanediol (EG + PRO; n = 96), blastocysts were placed in 10% EG plus 10% PRO in holding medium for 1 min and then transferred to a droplet of 20% EG plus 20% PRO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen. Both treatments were performed using the Cryotop system. Results were compared with embryos (n = 118) not submitted to cryopreservation. The evaluation was done by the hatching rate of blastocysts at Day 9, being higher (86.4%) for embryos not cryopreserved, when comparing with 77.1% for group EG + PRO and 72.9% for group EG + DMSO (P < 0.05). In the second experiment, Day 5 embryos obtained in vitro from Nelore donors were cultured using SOF medium with 10 μM forskolin (n = 112) or not (control; n = 101), being all submitted to cryopreservation using Cryotop and the same vitrification method for group EG + DMSO. Results were compared with embryos cultured with SOF medium and not submitted to cryopreservation (n = 96). The evaluation was performed by considering hatching rate at Day 9, being higher (85.4%) for not cryopreserved, when compared with 63.3% for control and 70.5% for forskolin group (P < 0.05). Considering embryos submitted to cryopreservation, the hatching rate was higher (P < 0.05) for the forskolin group.
115 ADDITION OF ETHANOL AT SUB-STRESS CONCENTRATIONS DURING IN VITRO MATURATION OF BOVINE OOCYTES IMPROVES BLASTOCYST CRYOTOLERANCE
Authors: Shehab-El-Deen M. A. M. M. , Leroy J. L. M. R. , Maes D. , Van Soom A.
Published: August 2010

In in vitro experiments, the percentage of bovine oocytes that develops to the blastocyst stage is much lower compared with the in vivo counterparts. The quality of the oocyte is the main factor affecting blastocyst yield. Moreover, in vitro-produced bovine embryos are more sensitive to cryo-injuries than those produced in vivo. Exposure of oocytes to sub-lethal concentrations of stressors may enhance their quality through upregulation of intracellular shock proteins. We aimed to evaluate whether addition of ethanol at low concentrations (0.27 or 0.53%) during oocyte maturation could have a carry-over effect on embryo quality and could subsequently affect embryo cryotolerance. Cumulus-oocyte complexes (n = 934) were matured in serum-free TCM199 plus 20 ng mL-1 of epidermal growth factor (control), supplemented with ethanol 0.27% (treatment 1) or 0.54% (treatment 2), in 3 replicates. After fertilization, the presumptive zygotes were cultured for 6 days in modified SOF medium supplemented with 5% fetal calf serum (FCS); the number of blastocysts was recorded and classified. Then, expanded blastocysts were cryopreserved by open pulled straw vitrification using the 2-step approach described by Vajta G et al. (1998 Mol. Reprod. Dev. 51, 53-58). After 24 h, vitrified embryos were warmed and cultured in groups of <25 per 50 μL droplet of modified SOF medium with 5% FCS under mineral oil for 48 h and examined for re-expansion and hatching. Differences between the groups in blastocyst yield were analyzed by ANOVA; differences in survival rates between the groups were analyzed using logistic regression analysis. For all statistical models, group was included as fixed effect, and also the effect of replicate was included. Differences were considered to be statistically significant when P < 0.05. Addition of ethanol to in vitro maturation media had no significant effects on blastocyst yield on 7 dpi. Also, addition of ethanol at 0.27% did not affect blastocyst cryotolerance. However, addition of ethanol 0.54% to in vitro maturation media significantly increased the survival of bovine blastocysts after vitrification (P < 0.01; Table 1). The results of the present study indicate that maturation of oocytes under ethanol stress at low concentrations has carry-over effects on embryo quality, leading to improved cryotolerance.

Table 1.Survival percentage (mean ± SE) of vitrified expanded bovine blastocysts matured in the presence of sub-stress concentrations of ethanol

The authors thank I. Lemahieu and P. Vandamme for their excellent technical support. This research was supported by the special research fund, Ghent University (Grant, BOF/DOS No. 01W05706).
119 VITRIFICATION OF IMMATURE FELINE OOCYTES: EFFECTS ON ZONA PELLUCIDA ULTRASTRUCTURE AND DEVELOPMENTAL COMPETENCE
Authors: Tharasanit T. , Sananmuang T. , Manee-In S. , Adirekthaworn A. , Lohachit C. , Techakumphu M.
Published: August 2010

Oocyte cryopreservation has been used to restore fertility problems in women undergoing oocyte-toxic chemotherapy and also to salvage the genetic potential of endangered/valuable species. In domestic cats, knowledge regarding the cryopreservation technique of feline oocytes is rather limited. We examined the effect of OPS vitrification on zona pellucida ultrastructure and also the developmental competence of immature cat oocytes. Immature cat oocytes were vitrified using different CPA and CPA exposure techniques: (1) 2-step DMSO, n = 71; (2) 4-step DMSO, n = 78; (3) 2-step EG, n = 73; (4) 4-step EG, n = 117; (5) 2-step EG + DMSO, n = 67 and (6) 4-step EG + DMSO, n = 88. A combination of EG and DMSO or EG alone using 4-step equilibration technique yielded the highest maturation rates (28/88: 31.8% and 44/117: 37.6%, respectively). This was, however, significantly lower than nonvitrified controls (36/59, 61% maturation rate). We further examined the effect of OPS vitrification on ultrastructure of the zona pellucida using scanning electron microscopy and also to test the quality of the zona pellucida using sperm binding assay. Our modified OPS protocols did not significantly affect the appearance of zona pellucida of vitrified oocytes and also the number of tightly bound sperm as compared with controls (80.8 ± 63.8 v. 81.6 ± 70.4, respectively). Vitrification of immature oocytes significantly reduced the number of oocytes reaching cleavage and blastocyst stages compared with nonvitrified controls (24.8 and 7.5% v. 62.5 and 30.8% for cleavage and blastocyst formation rates, respectively). It is concluded that modification of OPS vitrification can be successfully applied for immature cat oocytes, in terms of zona pellucida ultrastructure, ability to bind sperm, and also their developmental competence.

This study was financially supported by MRG4980108, CHE-TRF Senior Research Scholars RTA-5080010, The Thailand Research Fund, and The Research Unit of Reproductive and Biological Science, Chulalongkorn University.
122 THE SURVIVAL RATE AND EMBRYONIC QUALITY OF BOVINE PARTHENOGENETIC BLASTOCYSTS POST-VITRIFICATION CRYOPRESERVATION
Authors: Zhang P. , Wang Z. W. , Tang B. , Zhang J. B. , Li Z. Y.
Published: August 2010

Cryopreservation of oocytes and embryos is very useful to conservation of animal genetic resources. Recently, parthenogenesis has received considerable attention as a tool for the production of stem cells. Oocytes and embryos undergo considerable morphological changes and functional damage during cryopreservation, and the survival rate is highly depending on species and developmental stage of oocytes and embryos. The aim of this study was to investigate the survival rate and embryonic quality of bovine parthenogenetic blastocysts post-vitrification cryopreservation. Cumulus-oocyte complexes (COC) from slaughterhouse ovaries were aspirated from 2-mm to 8-mm visible follicles with a 5-mL syringe. The COC were matured in vitro for 22 h in bicarbonate-buffered TCM199 media supplemented with 1 mg mL-1 of FSH, 10 mg mL-1 of LH, 1 mg mL-1 of 17-βiestradiol, and 10% FBS. After in vitro maturation, cumulus cells were removed from COC, oocytes with first polar body were activated by 5 μM ionomycin for 5 min and 2 mM 6-DMAP for 4 h. Subsequently, oocytes were co-cultured with bovine fetal fibroblast cells in SOF media supplemented with amino acids (1% NEAA and 2% EAA), 4 mg mL-1 of BSA, and 10% FBS at conditions of 38.5°C and 5% CO2 for 7 to 9 days. The good expanded blastocysts were selected and refrigerated in different vectors [glass micropipettes (GMP) and straws] and same vitrification solution (VS, 20% EG + 20% DMSO). Blastocysts were exposed to VS, loaded on vectors, and plunged into liquid nitrogen within 25 s. After two days refrigeration, vitrified blastocysts were thawed in air for 10 s and placed into 0.25 M sucrose solution for 1 min and 0.15 M sucrose solution for 5 min. Then, the blastocysts were cultured in the SOF medium same as above. Our results showed that when VS was 20% EG + 20% DMSO, the hatching rate (65%) of blastocysts loaded into GMP was significantly higher (P < 0.05) than that (19%) of blastocysts loaded into straws post-vitrification. Meanwhile, vitrified and nonvitrified blastocysts were fixed and stained for differential cell counting as described by Thouas GA et al. 2001 Reprod. Biomed. Online 3, 25-29). By the differential staining, the total cells of nonvitrified parthenogenetic bovine blastocysts were 102.7, which was higher (P > 0.05) than that (86.7) of vitrified blastocysts. Also, no significant difference (P > 0.05) was seen on ratios between vitrified blastocysts (ICM/TE = 0.22) and nonvitrified blastocysts (ICM/TE = 0.25). Our results indicated that a glass micropipette vector was much better than a straw in vitrification cryopreservation of bovine parthenogenetic blastocysts and caused less damage to blastocyst cells. This study lays the foundation for further research to increase the survival rate of vitrification cryopreservation of bovine embryos.

This work was supported by the grant from national support plan, China, No. 2007BAD55B03; corresponding author: Ziyi Li.
13 USE OF A NEW INTRAVAGINAL DEVICE TO RELEASE PROGESTERONE IN FIXED-TIMED ARTIFICIAL INSEMINATION IN NELLORE COWS (BOS TAURUS INDICUS)
Authors: Braga F. A. , Pardo F. J. D. , Miguez P. H. , Kehrle A. , Pimentel J. R. V. , Rocha C. C. , Madureira E. H.
Published: August 2010

Artificial insemination results can be improved using fixed-timed AI (FTAI). Currently, progesterone release devices, indispensable in FTAI procedures, such as DIB® (Schering-Plough, Kenilworth, NJ, USA), are made of silicone, a nonbiodegradable biocompatible polymer. Recently, biodegradable progesterone (P4)-releasing devices made of poly(3-hydroxy)butyrate-valerate copolymer (PHB-V) and poly-ϵ-caprolactona (PCL) have been developed. To investigate the efficiency on pregnancy rate of FTAI programs using a new intravaginal device made of PHB-V (Progestar®, Innovare, São Paulo, Brazil), 240 Nellore (Bos indicus) cows, between 3 and 8 years of age, were randomly assigned to 2 experiments. In experiment 1, new P4 devices DIB (control group, DIB1; n = 60) were compared with PHB-V (Progestar1; n = 76); in experiment 2, previously used DIB (DIB2; n = 52) and PHB-V (Progestar2; n = 52) devices were compared. All groups were treated with 2 mg of estradiol benzoate i.m. (Innovare) and received the intravaginal device according to group (Day 0). Together with P4 device withdrawal (Day 8) all animals received 300IU of eCG i.m. (Novormon®, Schering-Plough) and 150 μg i.m. of D-cloprostenol (Croniben®, Biogenesis-Bagó, Buenos Aires, Argentina). On Day 9, all cows were treated with 1 mg of estradiol benzoate and the FTAI was performed 30 h later. The data were submitted to ANOVA (PROC GLM of SAS, SAS Institute, Cary, NC, USA), separating as causes of variance the effect of treatment, postpartum period (PPP1-45 until 70 days and PPP2-71 until 90 days), body condition score (BCS4, BCS5, and BCS6 using a 1 to 9 scale), cyclicity (cycling and noncycling) and clean-up bulls (5 bulls nominated with numbers 1 to 5). Significant differences were considered to have P < 0.05. In experiment 1, interactions were not significant, there was no effect of treatment on FTAI (DIB1: 66.7% v. Progestar1: 65.8%) or breeding season pregnancy rates (BSPR; DIB1: 95.0% v. Progestar1: 94.7%) and no effect of other variables on FTAI pregnancy rates (FTAIPR) and BSPR. In experiment 2 there was no effect of treatment on FTAIPR (DIB2: 61.6% v. Progestar2: 55.8%) and BSPR (DIB2: 94.2% v. Progestar2: 88.5%). As ANOVA indicated a significant effect of clean-up bulls on BSPR, Tukey test was used to split it up. A bull of the Progestar2 group had lower performance (41.7%) causing the difference (albeit nonsignificant) on BSPR. This experiment showed that Progestar and DIB had the same efficiency on pregnancy rate and both treatments have the potential to produce FTAIPR >50% and BSPR >90%.

Fapesp-Fundação de Amparo à Pesquisa do Estado de São Paulo. CNPq-Conselho Nacional de Desenvolvimento Científico e Tecnológico.
134 THE DISTRIBUTION OF CYTOSKELETON AND CHROMATIN CONFIGURATIONS DURING EARLY EMBRYO DEVELOPMENT OF SWAMP BUFFALO (BUBALUS BUBALIS)
Authors: Techakumphu M. , Chankitisakul V. , Thaseephoo K. , Tharasanit T.
Published: August 2010

Microtubules and actin microfilaments have been demonstratedto be actively involved with fertilization and early embryo development. The objective of this study was to examine the redistribution of cytoskeleton and chromatin configurations in swamp buffalo oocytes through the initial cleavage event after in vitro fertilization. Sperm penetration was analysed at 6 h post IVF in 63 oocytes (3 replicates), whereas the chronology embryonic development in terms of the redistribution of cell cytoskeleton and chromatin configurations was studied in a total of 462 oocytes (7 to 8 replicates) at 12, 18, 24, 30, and 48 h after IVF. The oocytes were matured in vitro for 22 h. Then, IVF was performed as described previously (Totey et al. 1993). After fertilization, presumptive zygotes and embryos were fixed at various times (6, 12, 18, 24, 30, and 48 h) to examine spermatozoa penetration, redistribution of the cytoskeleton (microtubules and actin filaments), and chromatin configurations using epifluorescent microscopy. Staining was undertaken with wheat germ agglutinin to visualize the zona pellucida, monoclonal-α-tubulin-TRIT C to show the microtubules, 488 phalloidin to identify microfilaments, and DAPI to label the chromatin. At 6 h after fertilization, sperm penetration was observed in 44.4% of examined oocytes. At 12 h post IVF, maternal chromosomes of fertilized oocytes progressed to the second meiotic division and formed the female pronucleus simultaneously with the decondensation of paternal chromosomes. A dense network of microtubules was observed radiating from the base of the decondensing sperm head (referred to as sperm aster) At 18 h post IVF, the sperm chromatins became the male pronucleus. Simultaneously, the sperm aster increased in size and filled the whole ooplasm. The syngamy of the male and female pronuclei was completed by 24 h post IVF, which was associated with a dense array of microtubules. Cell cleavage was observed by 30 h post IVF. This was apparently facilitated by a dense network of actin microfilaments that formed in the middle of the dividing embryo. These results indicated that microtubules and actin microfilaments undergo changes after fertilization consistent with a crucial role during fertilization in swamp buffalo. The centrosomal material was paternally inherited.

This work was supported by TRF-MAG (MRG-WII515S056) and CHE-TRF Senior Research Fund (RTA5080010).
138 EFFECTS OF CLIMATIC HEAT STRESS ON EMBRYO MORTALITY OF GRAZING CATTLE IN AZORES
Authors: Geraldo A. C. A. P. M. , Souto L. R. , Moreira da Silva F.
Published: August 2010

Seasonal high temperatures and humidity negatively affects reproduction in cattle. This negative effect is caused by high body temperature, which influences endocrine status other than thyroid. In fact, heat stress is known to negatively affect ovarian function, the expression of estrus, the quality of oocytes, and embryo development. The aim of this study was, therefore, to relate the temperature and humidity index (THI) to the embryo mortality rate in grazing cattle in the Azores summer. For such purpose, the success of 2139 artificial inseminations in different breeds was evaluated from October 1, 2007 to March 31, 2009, performed by an artificial insemination company. The climacteric data were obtained by different meteorological stations located near the farms and were further transferred to the meteorological center. The embryonic mortality was calculated using the dates of artificial inseminations of the cows. Those who demonstrated estrus after 26 days, as observed by visual observation, were admitted as occurrence of embryonic death. For climatic values, relative humidity and temperature were recorded every hour and the THI calculated for each day between 1100 to 1700 hours. In the warmer months (July to September), the THI ranged between 68 and 79 (× = 73). In this period, THI was 18% higher than that observed in January to March, which ranged between 58 and 68 (× = 62). The embryonic mortality in summer was 51%, which was almost double that observed for the winter period. By linear regression analysis, a positive correlation between the monthly average values of the THI and the rate of embryo mortality was observed (R2 = 0.78). August was the most critical month and daily THI has always been up to 70, which is considered as the limit of cattle thermal comfort. The THI started decreasing in October (× = 72), but in this month embryo mortality rate was just like in August (57%). Results of the present study clearly demonstrated that in the conditions of the Azores, heat and humidity stress negatively affect the reproductive performance as evaluated by embryonic mortality. Although the THI decreased in October (× = 72), the rate of embryo mortality was still high (57%), which may have been a result of heat stress that the animals suffered during summer.

The first author is supported by the Regional Foundation for Science and Technology of the Azores Government. This study was supported by the IBBA Institute grant number M2.1.2/I/022/2008. CITA-A is fully acknowledged.
148 EFFECTS OF Wnt3A SUPPLEMENTATION ON BOVINE BLASTOCYST CELL NUMBER AND ALLOCATION
Authors: Goissis M. D. , Ross P. J. , Cibelli J. B.
Published: August 2010

Derivation of true bovine embryonic stem cells (ESC), as defined by their capacity to form robust teratomas and/or contribute to the germ line in chimeras, has not been achieved despite several attempts. It is possible that failures to derive bonafide bovine ESC are due to the inability of bovine embryonic cells to adapt to in vitro culture conditions that favor ESC derivation. Wnt pathways are involved in pluripotency and self-renewal of mouse and human ESC. Wnt signaling is also required for implantation competence in mouse blastocysts. Given the shared developmental potential between inner cell mass (ICM) and ESC, we hypothesized that Wnt could act on the ICM of bovine embryos increasing its proliferation potential. The objective of this study was to evaluate the effect of post-embryonic genome activation Wnt3A supplementation on blastocyst formation and cell allocation to ICM and trophectoderm (TE). In vitro fertilized bovine embryos at Day 4 of culture in KSOM medium were divided into 3 treatments: Control, no co-culture; co-culture with regular mouse embryonic fibroblasts (MEF); and co-culture with mouse L fibroblasts overexpressing Wnt3A protein (L-Wnt3A, Willert et al. 2003 Nature 423, 448-452). Embryos were cultured until Day 8 when blastocyst and hatching rates were recorded. Then, embryos were submitted to differential staining of ICM and TE by brief exposure to 0.25% Triton X-100 in PBS and staining with bisbenzimide and propidium iodide. Six IVF replications were performed and a total of 39 embryos were counted: 11 for Control, 16 for MEF, and 12 for L-Wnt3A. Only intact embryos after processing were used for cell count. Statistical analysis was performed by ANOVA using PROC MIXED of SAS software (SAS Institute Inc., Cary, NC, USA) in which each IVF was considered as a block with Tukey’s adjustment for mean comparison of rates and Bonferroni adjustments for mean comparison of cell counts. Results for blastocyst rate, hatching rate, ICM, TE, and total cell number are presented in the table below. Different superscript letters within columns indicate significant statistical difference (P < 0.05). These results indicate that L-Wnt3A fibroblast co-culture exerts a positive effect on bovine embryo cell number, resulting in a larger number of ICM cells in bovine embryos, which could be beneficial for ESC derivation attempts.

Table 1.Blastocyst and hatching rates, ICM, TE, and total cell number results
153 IN VITRO CULTIVATION OF CAPRINE EMBRYOS PRODUCED IN VIVO
Authors: Martinez A. C. , Tramontini R. C. M. , Saito Jr M. A. , Abreu C. O. , Alcalde C. R.
Published: August 2010

Independent of embryo stage, when grade III embryos are transferred, they usually provide poor pregnancy rates. The aim of this study was to compare the effect of different in vitro cultivation media on the development of in vivo derived grade III caprine embryos. Twelve adult crossbreed goats from the Animal Breeding and Reproduction Laboratory of Maringá State University were used for this experiment. The goats were not pregnant or lactating. After embryo collection, embryos were classified regarding their development and quality, according to the International Embryo Transfer Society manual. Morulae classified as grade III were used in this experiment. The embryos were cultivated in 2 different media: Holding Plus medium (Bioniche Animal Health, Belleville, Ontario, Canada) or PBS plus 10% of bovine fetal serum (Nutricell, Campinas, Brazil) in 38.5°C waterbath with water circulation for 24 hours. After 24 h, the embryos were morphologically assessed. The percentage of embryos that developed during the cultivation period was calculated to evaluate the effectiveness of culture media. The embryos that produced new cell divisions and changed from grade III (poor quality) to grade II or grade I (excellent or good) were considered developed embryos. Chi-square was used to determine statistical differences between media. In the present work, the rate of embryos that developed in Holding Plus medium (Bioniche Animal Health) was 75%, and with PBS plus 10% bovine fetal serum was 40% of the total cultivated embryos.

Table 1.Embryo development assessment after in vitro cultivation process
164 DEVELOPMENT OF MURINE EMBRYOS AFTER INJECTION OF UNCOATED GOLD AND SILVER NANOPARTICLES
Authors: Taylor U. , Garrels W. , Petersen S. , Barcikowski S. , Klein S. , Kues W. , Lucas-Hahn A. , Niemann H. , Rath D.
Published: August 2010

Novel techniques such as ultrashort pulsed laser technology to produce in situ bio-conjugated nanoparticles (NP) as biomarkers for optical imaging and intervention hold great potential to observe dynamic processes in gametes and embryos without affecting their developmental potential. With regard to toxicology, current preliminary evidence is mainly based on specific cell lines and suggests low toxicity for gold NP (GNP), but a higher toxicity for silver NP (SNP), which also possess a considerable antibacterial effect. Little is known about their impact on sensitive biological systems as early mammalian embryos. The present study investigated the potential toxicity of GNP and SNP in murine embryos for the first time. Since the NP were laser-generated, they formed a stable dispersion in water without the need of surface coating, which might have caused additional toxic effects. Approximately 10 pL of a NP díspersion (average NP diameter of 15 nm) containing 50 μg mL-1 of either GNP or SNP were injected into 1 blastomere of 2-cell-stage embryos (n = 93 or 75, respectively), derived from superovulated NMRI mice. Embryos injected with water alone (n = 79) served as controls. Untreated embryos (n = 92) were run as a handling control. Successful NP injection was confirmed using laser scanning confocal microscopy. After treatment, embryos were cultured for 72 h at 37°C and 5% CO2 in KSOM plus 1% BSA. Embryo development was assessed on a daily basis. No abnormal development was observed. The handling controls reached a blastocyst rate of 77.2%. A total of 66.2% of the water-injected embryos developed to blastocysts, compared to 62.4% and 56.0% after injection of GNP and SNP, respectively. Neither 1-way ANOVA nor an additional chi-square test indicated significant differences between treatment groups. In conclusion, these preliminary data indicate that intracytoplasmatic injection of GNP and SNP had no significant impact on embryo development. Further tests, including qRT-PCR of development-relevant genes in blastocysts and transfer of injected embryos into recipient animals to study potential long-term effects, are underway to gain a better understanding of GNP and SNP embryo toxicology.

We gratefully acknowledge the support of the Masterrind GmbH Verden and the NBank Niedersachsen.
165 OVARIAN RESPONSE AND EMBRYO RECOVERY RATES OF OLD MARES TREATED WITH A LOW DOSE OF EQUINE PITUITARY EXTRACT
Authors: Bonin B. F. , Dellaqua Jr J. A. , Alvarenga M. A.
Published: August 2010

Mares older than 15 years have low embryo recovery rates, mostly because of disturbances in follicle or oocyte maturation and early embryonic development. A recent publication showed that circulating LH level is lower in older mares (Ginther et al. 2009 Theriogenology 5, 780-788). Equine pituitary extract (EPE) is rich in LH and consequently is able to increase circulating LH levels after injection into mares. The present study aimed to evaluate the effect of low doses of EPE on follicular growth, ovulation rate, and embryo recovery rates in older mares. Embryo donors (n = 20) were Quarter Horses from a commercial embryo center that ranged in age from 15 to 20 years. Donors were used during 3 consecutive cycles. The first and third cycles were used as control cycles, and the EPE treatment was performed on the second cycle. During estrus in each of the 3 cycles, mares were inseminated with cooled or fresh semen from the same stallion and received an injection of deslorelin (1 mg i.m.) upon detection of at least 1 follicle of 35 to 40 mm in diameter. Embryo flushes were performed on Day 8 post-ovulation and were followed by administration of prostaglandin. On the treated cycle, EPE injections (7 mg twice daily i.m.) were started on Day 8 after ovulation until a preovulatory follicle was observed. The percentage of mares with more than one ovulation was compared using Fisher’s test. The mean number of ovulations and embryos, as well as follicular diameter, was compared using Student’s t-test. The percentage of mares with more than one ovulation as well as the number of ovulations per cycle were higher (P < 0.05) in the EPE-treated cycle (65% of mares and 1.8 ovulations, respectively) than in the control cycles before (5% and 1.0) and after treatment (5% and 1.0). Also, the mean number of embryos recovered per cycle was higher (P < 0.05) in EPE-treated cycles (1.0) than in nontreated cycles (0.42). However, the embryo recovery rate per ovulation was similar (P > 0.05) between nontreated (0.4 embryo) and treated cycles (0.6 embryo). Pregnancy rates of the transferred embryos were also similar between embryos recovered on treated (14/20; 70%) and nontreated cycles (9/17; 55%). The daily growth of the dominant follicle was not altered by the EPE treatment. However, embryos recovered during treated cycles were more advanced in development than controls, with more embryos (P < 0.05) classified as morula or early blastocysts when recoverd during nontreated (58%) v. EPE-treated cycles (0%). Based on the results of the present experiment, we can conclude that EPE treatment was able to increase the reproductive efficiency of older embryo donor mares. The improvement in embryo recovery rates on EPE-treated cycles seemed to be more related to the increase in the number of ovulations per cycle than with an improvement in embryo viability.
17 EFFECT OF A PROGESTERONE INTRAVAGINAL DEVICE ON PREGNANCY RATE IN CROSSBREED DAIRY COWS UNDER TROPICAL CONDITIONS
Authors: Cardenas J. , Portillo G. E. , Lucena C. , Cabezas M. , Kowalski A. A.
Published: August 2010

The objectives of these experiments were to evaluate the effect of intravaginal progesterone device on crossbreed Holstein × Carora on pregnancy rate under 2 conditions: cycling and noncycling cows. The first experiment was conducted on 135 noncycling cows (45 ± 3 days postpartum) divided in 2 groups. The first group was treated with a timed AI procedure using a progesterone device for 9 days with an eCG injection on Day 6 and estradiol benzoate after progesterone device removal followed by AI 48 h later. The second group was not treated and they were checked until they came in heat and were inseminated. The second experiment was performed on 123 cycling cows and they were divided in 2 groups. The first group was submitted to a time AI treatment similar to that in experiment 1, and the second group was not treated and they were inseminated upon heat detection. Pregnancy rate on Day 120 postpartum was recorded and pregnancy rate was compared among the different proportion of Holstein (H) × Carora (C) crossbreed cows. For the first experiment, differences (P < 0.05) were observed between ¾ Carora ¼ Holstein (46.15%) compared with the other crossbreed groups (10% 7/8 H 1/8 C, 21.42% 5/8 H 3/8 C, 29.16% ½ H ½ C). In the second experiment, differences (P < 0.05) were observed between ¾ Carora ¼ Holstein (57.14%) compared with the other crossbreed groups (6.66% 7/8 H 1/8 C, 30.76% 5/8 H 3/8 C, 33.33% ½ H ½ C). For the variable pregnancy rate at 120 days postpartum, significant differences (P < 0.05) were observed in cycling cows (39.2%) treated with the progesterone device v. nontreated cows (21%). In noncycling cows, there were no differences between treated and nontreated cows. In conclusion, the use of progesterone device did not improve pregnancy rate in the noncycling cows, which could be related to the negative energy balance that cows undergo during the first part of gestation. However, the use of the progesterone device in cycling cows did improve the pregnancy rate compared with nontreated, cycling cows. We demonstrated that this dairy farm has a serious problem in detecting estrous, which was help by use of the progesterone device and timed AI. Also, the Carora blood included in dairy animals help them to perform better under tropical conditions and they can became pregnant much faster than animals with a high level of Holstein blood.
177 LIVE FOAL RATE: EFFECTS OF EMBRYO TRANSFER AND DONOR MARE AGE
Authors: McCue P. M. , Wall J. J. , Brink Z. A. , Seidel Jr G. E.
Published: August 2010

Embryo transfer is now common in the equine breeding industry, but limited data are available evaluating pregnancy outcome. The objectives of this retrospective study were to 1) compare live foal rates in mares carrying their own pregnancy with live foal rates following embryo transfer and 2) evaluate the effect of mare age on pregnancy outcome. Medical records for mares bred during the years 2004-2006 to carry their own pregnancies or bred to donate an unfrozen embryo to a young (≤15 years old) recipient mare were analyzed. Mares were included if they were confirmed pregnant at 16 days after ovulation and pregnancy outcome data were available. Pregnancy outcome data were compared by Fisher’s exact test (2-tailed) and PROC GLIMMIX of SAS (SAS Institute Inc., Cary, NC, USA). A live healthy foal was born from 116 of 134 (86.6%) mares carrying their own foal and 144 of 171 (84.2%) embryo transfer recipients (P > 0.1). The live foal rate for "carry own" mares ≤15 years of age, 89/100 (89.0%), was not different (P > 0.1) from those >15 years of age, 27/34 (79.4%). Recipients of embryos from mares >15 years old had a live foal rate of 41/48 (85.4%), which was similar to the live foal rate of embryos from mares ≤15 years of age, 103/123 (83.7%) (P > 0.1). A significant linear trend (P < 0.05) for an increase in pregnancy loss with increased age was noted when data for both groups were combined (Table 1). In conclusion, no significant difference was noted in pregnancy outcome between embryo transfer recipients and mares carrying their own pregnancies.

Table 1.Pregnancy loss rates for “carry own” or ET donor mares of various ages

Supported in part by the American Quarter Horse Association (Amarillo, TX, USA).
178 USE OF A NEW INTRAVAGINAL DEVICE TO RELEASE PROGESTERONE IN FIXED-TIME EMBRYO TRANSFER EMBRYO RECIPIENTS
Authors: Pimentel J. R. V. , Miguez P. H. P. , Kehrle A. , Madureira E. H. , Braga F. A. , Rocha C. C. , Pardo F. J. D.
Published: August 2010

Progestagen-releasing devices are commonly used for fixed-time artificial insemination and fixed-time embryo transfer (FTET). The aim of this study was to evaluate the efficacy of a new progesterone-releasing intravaginal device made of poly(3-hydroxy)butyrate-valerate copolymer (PHB-V) and poly-ϵ-caprolactone (PCL; Progestar®, Innovare, São Paulo, Brazil) to synchronize crossbredcows and heifers for FTET by determining ovulation, conception, and pregnancy rates. A total of 205 recipients ranging in age from 2 to 8 years (heifers, n = 77; non-suckled cows, n = 69; suckled cows, n = 59) were used on 3 different farms during February, March, and April 2008. The experimental design was a generalized block design (cyclicity, body condition score, postpartum interval, and animal category, i.e. heifers, suckled, or non-suckled cows), with 2 different synchronization treatments for FTET (Progestar® v. Crestar®). Cows were assigned to either Crestar® (norgestomet ear implants; Intervet Schering-Plough, Kenilworth, NJ, USA; n = 103) or Progestar® (PHB-V; n = 102) treatments. All groups were treated with 2 mg of estradiol benzoate i.m. (Innovare) and received the intravaginal or auricular device according to their group (Day 0). Together with P4 device withdrawal (Day 8), all animals received 400 IU of eCG i.m. (Novormon®, Schering-Plough) and 500 μg i.m. of PGF2α, (Sincrocio®, Ouro Fino, Brasil). On Day 9, all cows were treated with 1 mg of estradiol benzoate (Innovare) and the innovulation was performed on Day 17. Embryos were maintained at 37°C during the innovulation period. The percentage of usable recipients (AR) based on the presence of a functional CL on the day of embryo transfer, conception rate (numbers of pregnant recipients of the total number transferred; CR), and pregnancy rate (numbers of pregnant recipients of the total number synchronized; PR) were determined by ultrasonography 23 days after embryo transfer. Data were submitted to ANOVA (PROC GLM, SAS Institute Inc., Cary, NC, USA) and significant differences were considered to have a P-value of <0.05. None of the interactions were significant and there was no effect of treatment on AR (72.8% v. 83.3%), CR (47.9% v. 42.7%), and PR (34.7% v. 35.0%) for Progestar® v. Crestar® groups, respectively. This experiment showed that Progestar® was as efficacious as Crestar® to synchronize crossbred cows and heifers for FTET.

FAPESP-Fundação de Amparo à Pesquisa do Estado de São Paulo and CNPq-Conselho Nacional de Desenvolvimento Científico e Tecnológico.
18 EFFECT OF GnRH TREATMENT IN LH SECRETION IN INTACT AND OVARIECTOMIZED NELLORE HEIFERS
Authors: Cardoso D. , Guerra F. F. , Nogueira G. P.
Published: August 2010

The gonadotropin-releasing hormone (GnRH) and its analogues become control reproductive function in farm animals. The GnRH agonist (GnRHa), which is used in veterinary practice, has a longer half-life and higher affinity to GnRH receptors than the native decapeptide. GnRH or GnRHa effects depend on the species, dose, route, and the moment of administration. As Nellore is the main bovine breed in Brazil, corresponding to 70% of Brazilian herds, more studies on its reproductive physiology are needed. The objective was to study luteinizing hormone (LH) secretion in intact (I, n = 4) and ovariectomized (Ov, n = 4) prepubertal Nellore heifers (18 months) after gonadorelin acetate administration (25 μg, iv; Lecirelina, Gestran Plus, ARSA S.R.L., Buenos Aires, Argentina) from samples collected every 15 min for 10 h. LH was quantified by RIA, with sensitivity of 0.03 ng mL-1 and CV of 11%. Maximum LH amplitude was considered as the peak for each period. Peaks were identified as an increase 2 times higher than intraassay CV and involving at least 3 samples. The time for the highest peak occurrence was determined by GraphPad Prism program (Prism 3.00 for Windows, GraphPad Software, San Diego CA, USA). Results were compared by the ANOVA followed by t-test either paired or not using GraphPad InStat (3.06 version). After GnRH treatment, maximum LH peak amplitude was higher in Ov (12.48 ± 1.41 ng mL-1) (P ≤ 0.05) than in intact heifers (6.42 ± 1.30 ng mL-1), but there was no significant difference (P ≥ 0.05) on LH pulse frequency, with only one peak in both groups during the 10-h period. Although there was a small delay in peak occurrence in I (63.75 ± 21.48 min) compared with Ov heifers (45 ± 16.43 min), there was no difference (P ≥ 0.05) between groups. The results suggested a higher hypothalamus sensitivity to GnRH in absence of gonadal steroids or a higher LH storage during the hypergonatropism period.

Support by FAPESP (São Paulo, Brazil); fellowship from CAPES.
180 PREGNANCY LOSS (BETWEEN 30 AND 60 DAYS) IN HIGH-PRODUCING REPEAT-BREEDER HOLSTEIN COWS SUBMITTED TO EMBRYO TRANSFER
Authors: Rodrigues C. A. , Teixeira A. A. , Ferreira R. M. , Ayres H. , Ranieri A. L. , Freitas B. G. , Baruselli P. S.
Published: August 2010

Reproductive efficiency has a major impact on profitability of dairy farms. Pregnancy loss in cattle is one of the major impediments for adequate reproductive performance. The aim of the present study was to evaluate factors associated with pregnancy loss between 30 and 60 days of gestation in high-producing repeat-breeder (>4 services) Holstein cows submitted to embryo transfer (ET). A retrospective analysis was performed using data from 1 commercial dairy farm, located in Descalvado, Brazil (22°01′27″S; 47°53′19″L). Overall, 2096 ET were analyzed from 2004 to 2008. High-producing Holstein cows from the same herd were used as embryo donors. Embryos were produced by superovulation using the same protocol (8 decreasing injection of FSH) and were transferred, either fresh or frozen-thawed, to repeat-breeder Holstein recipients 6, 7, or 8 days after estrus (synchrony of Day 1, Day 0, or Day 1 with the donor, respectively). Statistical analysis was performed using PROC GLIMMIX of SAS (SAS Institute Inc., Cary, NC, USA). Variables included in the model were synchrony between recipient and donor at ET day, fresh and frozen-thawed embryos, stage of embryo development, and embryo quality. All cows were examined for pregnancy by ultrasonography 23 and 53 days after ET (30 and 60 days of pregnancy). There was no effect of year. The pregnancy loss was greater (P = 0.005) for fresh (22.9%, 141/615) v. frozen-thawed embryos (19.4%, 288/1481). Also, pregnancy loss was influenced (P = 0.002) by the synchrony between recipient and donor at ET day [Day -1:18.3% (53/290); Day 0: 18.9% (233/1234); D1: 24.9% (144/578)]. However, there was no difference in pregnancy loss when the stages of embryo development [morula: 20.7% (295/1428); early blastocyst: 18.9% (67/355); blastocyst: 23.0% (59/256); expanded blastocyst: 14.0% (8/57; P = 0.31)] and embryo quality [grade 1: 17.6% (78/443); grade 2: 20.1% (216/1073); grade 3: 23.3% (135/580); P = 0.31] were compared. In conclusion, pregnancy loss was greater in high-producing repeat-breeder Holstein cows receiving fresh embryos compared to frozen-thawed embryos and when Day 1 recipients were used compared to Day -1 and Day 0.

Agrindus S/A.
183 EXERCISE INFLUENCES EMBRYO QUALITY IN MARES
Authors: Smith R. , Vernon K. , Gibbons J. , Kelley D. , Anton J. , Mortensen C.
Published: August 2010

Athletic mares are commonly utilized in an embryo transfer regimen during the breeding season, which allows owners to continue to train and compete with these animals. Exercising mares in a hot and humid environment has proven detrimental to embryo recovery rates. However, the effects of exercise under milder temperatures have not been examined. Our goal was to compare embryo recovery rates and embryo quality scores of mares under mild environmental conditions of 2 differing exercise regimens v. a single control group (n = 5). One treatment group (full exercise) consisted of mares (n = 5) exercised 6 days a week throughout the duration of the project. The partial-exercise group (n = 5) was exercised throughout the duration of the project 7 days a week, with full rest from day of detected ovulation to the proceeding embryo collection attempt. This was to limit any deleterious effects exercise may potentially have on the mare and subsequent early developing equine embryo. Exercise resumed the day after an embryo collection attempt for the partial-exercise group. Exercised mares were given a 1-month training period prior to the beginning of this project and were moderately exercised for 30 min in a mechanical exercise pen (free exerciser). Control mares were managed similarly as the other groups, however were not exercised. Mares were bred via artificial insemination and embryos were nonsurgically collected 7 d post-ovulation. Mean environmental temperature during the duration of this study was 27.1°C with average 60% humidity. Immediately following completion of exercise, mares in the full exercise group had a mean temperature increase of 1.0°C and partial-exercised mares increased 0.9°C. Exercise had a significant effect on embryo quality (P < 0.05, Table 1) but did not influence embryo recovery rate. The mean embryo quality score for the control group was 1.1, whereas the full-exercised group was 1.85 and partial-exercised group was 2.5. It appears from these data that exercise has a negative effect on embryo quality, thus lowering the availability of transferable embryos. Embryos of lower quality (≥3) have been shown to result in poor pregnancy rates in horses. Additionally, rest from ovulation to an embryo collection attempt did not improve embryo quality.

Table 1.Embryo recovery rate and quality score of embryos from control and exercised mares
187 EFFICIENCY OF DIAGNOSIS OF CLINICAL AND SUBCLINICAL ENDOMETRITIS IN CATTLE EVALUATED BY HYSTEROSCOPY
Authors: Madoz L. V. , de la Sota R. L , Susuki K. , Heuwieser W. , Drillich M.
Published: August 2010

The objective of this study was to evaluate the efficiency of vaginoscopy and rectal palpation (palpable liquid in uterine horns) compared with hysteroscopy (presence of pus in the uterine lumen) for the diagnosis of clinical endometritis (CE), and hysteroscopy compared with endometrial cytology for the diagnosis of subclinical endometritis (SE) in postpartum dairy cows. Thirty Holstein cows between 20 and 35 days postpartum were examined for diagnosis of CE with a vaginal speculum, by rectal palpation, and by hysteroscopy; and examined for diagnosis of SE with hysteroscopy and endometrial cytology. Categorical data were analyzed with PROC CATMOD (SAS®, SAS Institute, Cary, NC, USA) and continuous data were analyzed with PROC GLM (SAS®, SAS Institute, Cary, NC, USA). Sensitivity, specificity, predicted value of a positive and negative result, and efficiency were calculated using Win Episcope® 2.0 software (Clive, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK). Prevalence of CE diagnosed by vaginoscopy was 27%, by rectal palpation was 23%, and by hysteroscopy was 13%. When hysteroscopy, the only tool for direct examination of the endometrium, was used as the gold standard for diagnosis of CE, vaginoscopy had 100% sensitivity, 85% specificity, and 87% efficiency, and rectal palpation had 75% sensitivity, 85% specificity, and 83% efficiency. Prevalence of SE diagnosed by endometrial cytology was 35.3%. When endometrial cytology was used as the gold standard for diagnosis of SE, hysteroscopy had 11% sensitivity, 92% specificity, and 59% efficiency. In conclusion, vaginoscopy had higher sensitivity than, and similar specificity and efficiency to, rectal palpation for diagnosis of CE. Conversely, hysteroscopy, although having high specificity, had low sensitivity and efficiency for diagnosis of SE. Hysteroscopy proved to be efficient for diagnosis of CE but inefficient for diagnosis of SE.

The authors gratefully acknowledge the support of the study by World of Medicine (WOM), Berlin, Germany, for providing all endoscopic equipment and the cooperation with the dairy farm in Brandenburg, Germany. Vanina Madoz visit to the Freie Universität Berlin was supported by a fellowship from the Deutscher Akademischer Austauschdienst, DAAD.