bluuebunny/crossref_metadata_2025 huggingface dataset:
116 TAUROURSODEOXYCHOLIC ACID PREVENTS ENDOPLASMIC RETICULUM STRESS-INDUCED APOPTOSIS IN PREIMPLANTATION PIG EMBRYOS
Authors: Kim J.-S. , Lee K.-S. , Song B.-S. , Zhang J.-Y. , Choo Y.-K. , Lee K.-K. , Koo D.-B.
Published: December 2008

Apoptosis is an important determinant for the normal development of preimplantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. The efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting classic pathways of apoptosis. Therefore, we explored the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis in preimplantation porcine embryos. Also, TM (tunicamycin; an ER stress inducing chemical reagent) was used to investigate the effect of ER-stress on pig embryo development. After in vitro maturation and fertilization, presumptive porcine embryos were cultured in NCSU23 medium supplemented with 200 μ g mL–1 TUDCA or 1 μg mL–1 (TM) for 6 days at 39°C, 5% CO2 in air. All data were analyzed by using Duncan test of ANOVA by Statistical Analysis System (SAS). When treated with TM during culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage compared with 27.4% (28/102) of the embryos in the control group (P < 0.05). We also confirmed that TM stimulates up-regulation of ER stress response genes, such as XBP-1 mRNA, and induces a high rate of apoptosis. Whereas the frequency of blastocyst formation in the TUDCA-treated group was increased compared with that in the control group (32.8%, 49/149 v. 22.2%, 32/144), P < 0.05). Furthermore, the blastocyst cell number was enhanced (30.6 v. 39.5) and apoptosis reduced (TUNEL positive nuclei number, 6.0 v. 3.2) by TUDCA treatment in pig embryos. As the result of real-time quantitative RT-PCR analysis, the expression of anti-apoptotic Bcl-xl gene was increased in the blastocyst stage by TUDCA treatment, whereas expression of pro-apoptotic Bax was decreased. In addition, we also confirmed that TUDCA decreases the rate of TM-induced apoptosis in preimplantation stage pig embryos. Our results indicate that TUDCA improves the developmental competence of porcine embryos by modulating the ER stress-induced apoptosis during preimplantation stage.
12 PHARMACOLOGICAL STIMULATION OF OVULATION IN THE BLACK FLYING-FOX (PTEROPUS ALECTO)
Authors: Melville D. F. , Crichton E. G. , O'Brien G. M. , Johnston S. D.
Published: December 2008

Artificial insemination (AI) involves the accurate detection of oestrus, the ability to pharmacologically induce ovulation, or both. This is particularly important in flying-foxes (Pteropus spp.) that do not have an overt behavioural oestrus. Pregnant mare serum gonadotropin (PMSG) stimulates folliculogenesis and ovulation in the Little Red Flying-fox (P. scapulatus; Towers PA and Martin L 1985 Proc. Aust. Soc. Reprod. Biol. 17, 115). In this study, a dose rate of 15 IU PMSG was used. Our study investigated if 15 IU PMSG would sufficiently induce ovulation in the larger, Black Flying-fox (P. alecto) or if a higher dose (30 IU) would be necessary. Before the mating season, a single injection of PMSG (Folligon®, Intervet Australia Pty Ltd, Bendigo East, Australia) was administered i.m. to eight adult females at 15 IU (n = 4) or 30 IU (n = 4) on Day 0. On Day 4, semen was collected by electro-ejaculation from adult males and inseminated intravaginally into females under isoflurane (Forthane®, Abbott Australasia Pty Ltd, Sydney, Australia) anaesthesia. Ovaries and reproductive tracts were surgically removed on Day 6, fixed, serially sectioned and stained using Gomori’s Stain. Histological sections were examined for evidence of ovarian activity and the presence of ova and spermatozoa in the reproductive tracts. Preliminary observations showed evidence of ovulation in both groups in the form of at least one CL in either ovary and an ovum in the ipsilateral oviduct or uterine horn. Ovaries of females stimulated with 30 IU PMSG differed noticeably from those treated with 15 IU in containing multiple, large, collapsed luteinized follicles with retained oocytes. Vascularization and glandular hypertrophy of the endometrium was also more evident in the higher dose group. No spermatozoa were observed in any of the excised tracts. These results suggest that whereas both doses of PMSG induce ovulation, the administration of 30 IU PMSG may over-stimulate the ovaries. This, in turn, could lead to an unphysiological environment for successful fertilization and embryonic development. A dose level of 15 IU or slightly above may be sufficient for subsequent attempts to stimulate folliculogenesis and ovulation in P. alecto. The absence of spermatozoa suggests that the site of insemination, the number of spermatozoa inseminated, or both requires further investigation if AI is to be successfully implemented in these species. We conclude that the ovarian responses to PMSG indicate that pharmacological induction of ovulation can be successfully achieved and thereby utilized in AI programs of endangered Pteropus species.
129 EFFECT OF DEVELOPMENTAL STAGE OF ICSI-PRODUCED EQUINE EMBRYOS ON PREGNANCY RATES
Authors: Stokes J. E. , Squires E. L. , Suh T. K. , Altermat J. L. , Carnevale E. M.
Published: December 2008

Intracytoplasmic sperm injection (ICSI) can be used to produce offspring from mares or stallions with fertility problems. Early embryos can be transferred into recipients’ oviducts or embryos can be cultured for nonsurgical transfer into recipients’ uteri. The aim of this research was to evaluate the optimal time to transfer ICSI-produced embryos into recipients’ uteri. The objective was to compare pregnancy rates after the nonsurgical transfer of early morulae, compact morulae, and blastocysts. Oocytes were collected by transvaginal, ultrasound-guided follicular aspirations between 20 and 24 h after administration of deslorelin (1.5 mg, i.v., Francks Pharmacy, Ocala, FL, USA) to donors. Oocytes were cultured for 16 to 18 h in M199 (Invitrogen, San Jose, CA, USA) with 10% FCS (HyClone, Logan, UT, USA), 0.2 mm pyruvate (Sigma, St. Louis, MO, USA) and 25 μg mL–1 gentamycin (Sigma, St. Louis, MO, USA) at 38.5°C and 6% CO2. Cumulus cells were denuded by gentle pipetting, after oocytes were placed into hyaluronidase (500 U mg–1, Sigma, St. Louis, MO, USA). Oocytes were injected with a single sperm from one of two stallions, with sperm being frozen or sex-sorted and refrozen (Squires EL et al. 2008). Forty-six of 62 (74%) injected oocytes cleaved. The presumptive zygotes were cultured in DMEM/F12 (Sigma, St. Louis, MO, USA) with 10% FCS at 38.5°C and an atmosphere of 5% CO2, 5% O2 and 90% N2. Embryos were placed in fresh medium every 3 days. Injected oocytes were observed for cleavage at 2 days, and embryos were assigned to a transfer group. Embryos were transferred as early morulae (EM, 8-cell to precompaction stage, n = 14), compact morulae (CM, postcompaction, n = 10) or blastocysts (B, observed blastocoele, n = 9) into recipients at 3 to 5 days (EM), 3 to 6 days (CM) or 5 to 6 days (B) after the recipient’s ovulation or follicle aspiration. Pregnancy scans were performed on Day 12, 14, and 16 after ICSI, and pregnant recipients were examined until 30 days to detect the embryo proper and heartbeat. Number of embryonic vesicles detected per transferred embryo was determined by Fisher’s Exact Test. Pregnancy rates differed (P = 0.0017) among groups (EM, 1/14, 7%; CM, 4/10, 40%; B, 7/9, 78%), with fewer (P = 0.001) EM than B resulting in embryonic vesicles; however, pregnancy rates were not significantly different between CM and other embryo stages. An embryo proper with heartbeat was observed for all pregnancies, with the exception of one pregnancy resulting from the transfer of a blastocyst. In this study, all blastocysts were transferred prior to the embryo attempting to hatch from the zona pellucida, but after the appearance of a distinct blastoceole. In our study, pregnancy rates were higher after the transfer of later v. earlier stages of embryo development.
13 SEASONAL CHANGES IN ION CONCENTRATION, ION-CHANNEL EXPRESSION, ACROSOME INTEGRITY, AND BOVINE SPERM QUALITY
Authors: Orgal S. , Roth Z.
Published: December 2008

The decrease in dairy cow fertility during the summer is mainly associated with the deleterious effects of heat stress on the female reproductive tract. The present study suggests that the decreased reproductive performance is, in part, a result of using semen of inferior quality. Evaluated parameters included (1) ionic concentrations in the seminal fluid of bull semen, including [Ca2+], [K+], and [Na+]; (2) expression of ion channels CNGB1, CNGA3, and IP3R; (3) parameters of semen quality such as volume, concentration, motility, and progressive motility; and (4) acrosome integrity. Semen was collected from 5 representative bulls throughout the summer (August and September) and winter (December and January), and was evaluated according to a computerized sperm quality analyzer for bulls (SQA-Vb, Medical Electronic Systems, Caesarea, Israel). [Ca2+], [K+], and [Na+] in the seminal fluid were determined by inductively coupled plasma-atomic emission spectrometry. A semiquantitative PCR and the computer program Scion Image (Scion Corporation, Frederick, MD) were used to determine RNA expression of ion channels CNGB1, CNGA3, and IP3R. Acrosome integrity was assessed by a triple-fluorescence test, which included Hoechst 33342 (h33342), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and propidium iodide (PI) staining. Data were presented as mean ± SD and analyzed by t-test (JMP-in 5.1). Findings revealed seasonal differences in [K+], [Na+] (P < 0.01), and [Ca2+] (P < 0.08) in the seminal fluid. These were associated with differences in RNA expression for ion channels CNGB1, CNGA3 (P < 0.01), and IP3R (P < 0.09), which are known to be involved in acrosome reactions. Although no differences were found in fresh semen, a progressive decrease in motility was noted for post-thaw semen collected in the summer (P < 0.05). Furthermore, the proportion of sperm cells with damaged acrosomes was higher in post-thaw semen collected in the summer than in its counterpart collected in winter (54.2 ± 3.5% v. 51.4 ± 1.9%, respectively; P < 0.08). The results suggest that semen collected during the summer is less able to survive cryopreservation, as reflected by its inferior vitality post-thawing. Further examination is required to determine whether such alterations are involved in the low summer fertility of dairy cows.

The authors thank the Israeli Artificial Insemination Center, Sion, for semen and Medical Electronic Systems (Caesarea, Israel) for providing the SQA-Vb sperm quality analyzer.
138 GENETIC PARAMETER ESTIMATION FOR EMBRYO TRANSFER TRAITS
Authors: Di Croce F. A. , Saxton A. M. , Rohrbach N. R. , Schrick F. N.
Published: December 2008

Genetic selection has made tremendous progress on economically important traits in the beef industry. Most of the progress has been from quantitative genetics through use of expected progeny differences (EPD). These values allow prediction of differences in progeny of a sire compared to progeny of other sires. Development of EPD for male and female reproductive traits has largely been ignored because of low heritability of reproductive traits, even though reproduction plays a vital role in the economics of beef operations. Therefore, continued research in the area of genetic selection for fertility is becoming increasingly important. Critical limiting factors for animal breeding programs using MOET nucleus schemes include variability in superovulatory response of donor animals and resulting pregnancy of transferred embryos. Thus, the overall objective of this research was to develop genetic parameters associated with MOET to assist producers in identifying animals with greater genetic merit for these protocols. Records were examined from a large-scale MOET system in beef cattle that contained data only for cows in which at least one transferable embryo was obtained. Data on these animals were extracted and analyzed on 10 425 transferred embryos (2900 collections) from 611 donor animals (Angus, Brangus, and Charolais) utilizing semen from 215 bulls. Phenotypic traits examined included pregnancy status of the recipient following transfer (ET-preg; determined by rectal palpation at 60 days post-transfer and/or confirmed calving date of recipient), number of transferable embryos per collection (ET-trans), and number of unfertilized ova at collection (ET-UFO). Basic statistical analysis and pedigree/trait files were developed using procedures in SAS (SAS Institute, Cary, NC). Genetic parameters were estimated for a single-trait animal model using restricted maximum likelihood (REML) procedures in Wombat (Meyer K 2007 Zhejiang Uni. Science B 8, 815–821). Wombat also computed EPD and standard errors for each trait evaluated. The model included fixed effects of year as well as random animal and residual effects. The EPD for ET-preg ranged from –6.1 to 4.4% (SE = 2.2 to 4.2) for semen sires (sires of the transferred embryos) and –5.3 to 3.8% (SE = 3.2 to 4.2) for donor animals. Additionally, the heritability estimated for ET-preg was 0.03. Heritability estimated for ET-trans was 0.00, indicating minute genetic variation and thus, EPD were not presented. Heritability estimated for ET-UFO was 0.05 with EPD values (deviation of the number of UFO from the mean) ranging from –0.6 to 0.8 (SE = 0.3 to 0.6) for semen sires and –0.4 to 1.1 (SE = 0.5 to 0.6) for donor cows. As previously shown for reproductive traits, heritability of ET-preg, ET-trans, and ET-UFO was low. Genetic improvement in fertility by selection on embryo transfer traits is possible, but progress would be slow. Further studies are underway on a larger dataset to refine these estimates and to examine repeatability.
15 FERTILITY LEVELS IN EWES TREATED WITH MEDROXIPROGESTERONE ACETATE OR FLUOROGESTONE ACETATE SPONGES PLUS EQUINE CHORIONIC GONADOTROPIN IN DIFFERENT PHASES OF THE OESTRUS CYCLE
Authors: Requena F. , Escribano B. , Tovar P. , Gardón J. C.
Published: December 2008

Commercial programs of estrus synchronization in ewes are performed with intravaginal sponges impregnated with synthetic progestagens such as medroxiprogesterone acetate (MAP) or fluorogestone acetate (FGA). Normally, sponges are placed deep into the vagina in cyclic females at different points in the estrus cycle. The aim of the present study was to compare fertility levels in ewes with estrus synchronized by using commercial sponges impregnated with either 60 mg of MAP (eMAP) or 30 mg of FGA (eFGA), placed during the follicular or luteal phase of the estrus cycle, plus eCG. A total of 273 sexually mature Merino ewes belonging to the same herd, aged between 2.5 and 5.5 years and clinically healthy, were scanned by transrectal ultrasonography with an 8-MHz probe (Aquila, Esaote-Pie Medical, Maastricht, The Netherlands) to visualize the ovarian status in the reproductive season. Ovaries without CL and follicles ≥4 mm were considered in the follicular phase, whereas ovaries with CL and follicles ≤4 mm were considered in the luteal phase. Thus, estrus synchronization was performed in 134 ewes treated with eMAP (Sincrogest, Ovejero Lab, Spain), of which 40 were in the follicular phase and 94 were in the luteal phase. In contrast, 139 ewes were treated with eFGA (Sincropart, CEVA Lab, Barcelona, Spain), of which 42 were in the follicular phase and 97 were in the luteal phase. Sponges were removed in all females after 12 days and 500 IU of eCG (Serigan, Ovejero Lab, Leon, Spain) was given. Ewes were then placed jointly with rams, at a ratio of 5 to 1, until no signs of mount were detected. Twenty-five days after the withdrawal of males, pregnancy rates were determined by transrectal ultrasonography with an 8-MHz probe. Fertility levels of synchronized estrus with the eMAP or eFGA sponges during the follicular or luteal phase are expressed as a proportion of ewes and were analyzed by a chi-square Pearson analysis. Significance was set at a level of P < 0.05. After ultrasound scanning of the initial 273 ewes, 30.04% were considered to be in the follicular phase, whereas 69.96% were in the luteal phase of the estrus cycle. Pregnancy rate for eMAP-treated ewes during the follicular phase was 67.50% and was 69.14% for treatments during the luteal phase. In addition, for ewes treated with eFGA, pregnancy rate was 73.80% during the follicular phase and 73.19% during the luteal phase. No significant differences were found in pregnancy rates for either eMAP- or eFGA-treated ewes during the follicular or luteal phase of the estrus cycle. In conclusion, these results suggest that during the breeding season, fertility levels were not affected in ewes whose estrus was synchronized with intravaginal sponges impregnated with MAP or FGA. In addition, pregnancy rates were not affected when the synchronization treatments began during the follicular or luteal phase of the estrus cycle.
162 REPRODUCTIVE CYCLES, PREGNANCY AND REVERSAL OF LONG TERM ACYCLICITY IN CAPTIVE SOUTHERN WHITE RHINOCEROS AT HAMILTON ZOO
Authors: Morrow C. , Kudeweh S. , Goold M. , Standley S.
Published: December 2008

In 1999, the New Zealand captive population of Southern white rhinoceros (Ceratotherium simum simum) was increased with the addition of six wild-caught founders. We report on the breeding success of two females (est. 5 to 6 years at import) and a female born in 2000 (conceived in the wild). Fecal samples were collected 1 to 3 times per week from females (n = 3) for several periods (5 to 24 months) between 2000 and 2008. Reproductive cycles and pregnancy were characterized using fecal progestogen concentrations, observations of courtship behaviour and parturition dates. The average length of the reproductive cycle was 31.6 ± 0.6 days (range 26–38 days, n = 34 cycles). Four pregnancies were confirmed in one female with a sex ratio of 1 female to 3 males (including 1 male loss at an estimated 5 months of gestation), known gestation lengths were 514 and 507 days. In the non-reproducing adult female, long cycles (67.2 ± 1.3 days, n = 5) were detected in addition to regular cycles (31.9 ± 0.9 days, n = 13) during the first 3 years in captivity, consistent with previous reports (Schwarzenberger F et al. 1998 An. Repro. Sci. 53, 173–190; Brown JL et al. 2001 Zoo Biol. 20, 463–486). The absence of breeding behavior and low progestogen concentrations for an extended 4-year period was cause for concern for the Australasian Species Management Programme managers (Hermes R et al. 2004 An. Repro. Sci. 82–83, 49–60; Hermes R et al. 2006 Theriogenology 65, 1492–1515). In 2007, the social structure of the Hamilton Zoo rhinoceros group was altered by exchanging the breeding male and two male offspring for a wild-caught male from Auckland Zoo. Within two months of arrival the long-term acyclic female had resumed cycling and had conceived six months after introduction of the new male. The young captive-born female continued to have regular length cycles and mating was observed with the new male. Recently, early embryonic loss was determined by ultrasound in the young female having a long 70 day cycle determined by fecal analysis. Fecal progestogen concentrations were useful for diagnosing pregnancy after 3 months gestation with concentrations four times higher than luteal phase concentrations by 9 to 12 weeks after mating (n = 3). Two females exhibited seasonal acyclic periods characterized by low progestogen concentrations corresponding to decreasing daylength (autumn and winter months). Fecal progestogen monitoring of reproductive status and pregnancy combined with behaviour observations has provided valuable information for breeding management decisions and planning for parturition in this population. The reversal of long-term acyclicity and a resulting pregnancy that represents two original founders is particularly significant for the genetic diversity of the Australasian population of white rhinoceros.
183 EFFECT OF WELL IN WELL CULTURE OF BOVINE EMBRYOS ON GENE EXPRESSION PROFILE
Authors: Ghanem N. , Hoelker M. , Phatsara C. , Schellander K. , Tesfaye D.
Published: December 2008

To culture embryos in small groups, the well in well culture system (miniwells harboring 1 single embryo within the well) has been developed previously. In this work, we aimed to examine the effects of the microenvironment provided by well in well culture and embryo density on the relative abundance of transcripts in the resulting embryos. Cumulus–oocyte complexes (COCs) were aspirated from small follicles (2 to 8 mm), and COCs were cultured in 400 μL of modified TCM (TCM-199, Sigma, Taufkirchen, Germany) supplemented with 12% heat-inactivated estrous cow serum and 10 μg mL–1 of FSH (FSH-p, Sheering, Kenilworth, NJ, USA) for 24 h at 39°C in a humidified atmosphere with 5% CO2 in air. Fertilization was performed in Fert-TALP supplemented with 1 μg mL–1 of heparin. Zygotes were allocated randomly in 2 groups, namely: well in well culture (16 miniwells of 0.7 diameter and deepness each containing 1 embryo per well) and group of 16 (group culture of 16 embryos per well). Six pools each containing 20 Day 7 blastocysts derived from the first 2 groups were used to investigate large-scale gene expression analysis using BlueChip cDNA-Array. Three pools each containing 5 blastocysts were used for Array data validation by real-time PCR using primers specific to 5 selected genes (ATP5, PLAC8, KRT8, S100A10, and ZP3). During validation in vivo-derived bovine blastocysts were included to be used as standard. Significance Analysis of Microarray identified 75 transcripts differentially expressed between the 2 groups. Blastocysts derived from well in well culture were found to be enriched with genes regulating different molecular functions including structural constituent of ribosome (RPS29), protein binding (Cul1), calcium ion binding (S100A10, NPTX2), nitric oxide synthase regulator activity (HSPCA), and RNA polymerase II transcription factor activity (UHRF1). However, blastocysts derived from group of 16 culture were found to be enriched with genes involved in oxidoreductase activity (ALOX15, AKR1B), cytochrome-c oxidase activity (COX7A2), hydrogen ion transporting ATP synthase activity (ATP5O), transcription (PTTG1), and cell redox homeostasis (TXN). According to their biological process, genes enriched in blastocysts derived from well in well culture belong to small molecule transport and signal transduction, whereas most downregulated genes have a metabolic function. Comparison of the transcript abundance of the 5 selected genes in the 3 embryo groups showed that the expression of ATP5, PLAC8, and KRT8 in embryos from well in well culture resembles to the relative abundance in blastocyst derived from in vivo culture. However, with respect to the expression of S100A10 and ZP3 genes, blastocysts derived from group culture showed similarity with embryos derived from in vivo. In conclusion, microenvironment affects the gene expression pattern of the resulting embryos.
186 DUODENAL AND RENAL TRANSIENT RECEPTOR POTENTIAL VANILLOID 6 APPEAR TO BE DISTINCTLY REGULATED BY SEX STEROID HORMONES, ESTROGEN AND PROGESTERONE, IN IMMATURE FEMALE RATS
Authors: Kim J.-H. , Choi K.-C. , Jeung E.-B.
Published: December 2008

Calcium-related proteins include transient receptor potential vanilloid (TRPV) 5 and 6, plasma membrane calcium-ATPase 1b (PMCA1b), and calbindin-D9k and -D28k. The TRPV6 is a major calcium channel located in the apical and basolateral membranes of cell and distributed widely in many other organs, especially in the exocrine tissues such as intestine and uterus. TRPV6s are generally regulated by vitamin D, a dietary calcium ion and hormone. In particular, uterine TRPV6 appears to be affected by sex steroid hormones, which are altered according to estrous cycle and pregnancy. In order to discover the effect of sex steroid hormones on the regulation of TRPV6, we examined the expression of TRPV6 mRNA by using RT-PCR and real-time PCR, and protein expression of TRPV6 by immunohistochemistry (IHC) in the uterus, duodenum, and kidney. To evaluate the effect(s) of sex steroid hormones on its uterine, duodenal, and renal regulation, 17β-estradiol [E2; 40 μg kg–1 of body weight (bw)] and/or progesterone (P4; 4 mg kg–1 of bw) or vehicle (n = 6/each group) were subcutaneously injected into Sprague-Dawley immature female rats (14 days old, n = 24 in total) for 3 days. As a result, the treatments of immature rats with E2 or P4 increased TRPV6 mRNA for calcium function or regulation in the uterus of immature rats. To confirm the specificity of E2 or P4 through their receptors, we treated the immature rats (extra n = 24 in total) with an estrogen receptor-antagonist, ICI 182,780 (ICI; 30 μg kg–1 of bw), and/or progesterone receptor antagonist, RU 486 (10 mg kg–1 of bw), at 3 days prior to E2 or P4 injection. Consequently, an increase in TRPV6 mRNA was observed in the following 2 treatments; ICI plus E2/P4 and E2/P4 alone. In IHC, we further observed that the expression of duodenal TRPV6 was increased by E2 or P4 and E2 or P4 plus ICI, while no difference was observed in renal TRPV6 by the treatments of sex steroid hormones. In conclusion, these results indicate that the expressions of uterine and duodenal TRPV6 may be induced by E2 and P4, but its renal expression may not be controlled by these steroids.
203 EFFECT OF FRUCTOSE ON THE DEVELOPMENT OF BOVINE SEXED EMBRYOS IN VITRO
Authors: Chaubal S. A. , Xu J. , Yang X. , Du F.
Published: December 2008

It is important to optimize the condition of culture to promote the development of embryos fertilized by sex-sorted sperm. Fructose is a monosaccharide hexose that can be used as a substrate for glycolysis. Recently, there has been interest in exploring the embryotrophic properties of fructose during bovine embryo culture. The present study was undertaken to ascertain the effect of supplementation of fructose in culture on the development of sexed embryos in vitro. A total of 2310 slaughterhouse dairy oocytes, spread over 3 experimental replicates, were subjected to routine in vitro maturation (IVM) for 22 h. The maturation medium was M-199 with Earl’s salts, L-glutamine, 26.19 mm sodium bicarbonate, and 25 mm HEPES plus 10% fetal bovine serum. Fertilization of IVM oocytes was performed in Brackett and Oliphant’s medium by using frozen and sorted X-sperm from a single bull. Groups of 50 matured oocytes were added to a final volume of 100 μL of fertilization medium, with a final concentration of 0.3 × 106 spermatozoa mL–1. The oocytes were incubated with X-sperm for 6 h in 5% CO2 humidified air at 39°C. Presumptive zygotes were randomly allocated to culture either in a control medium (6 mg mL–1 of BSA CR1aa medium) or with 4.5 mm fructose added to CR1aa culture medium for 7 days in an atmosphere of 5% O2, 5% CO2, and 90% N2 at 39°C. Embryo cleavage, and development to the morulae and blastocyst stages were evaluated on Days 2, 5, and 7 of culture, respectively. Embryos were graded according to International Embryo Transfer Society standards. The total number of blastocysts recorded was the sum of grade C1 and C2 embryos. Data were analyzed by using the General Linear Model (SPSS 11.0, SPSS Inc., Chicago, IL, USA). As shown in Table 1, addition of fructose to the culture medium affected neither the rate of cleavage nor subsequent development to the morula stage or total blastocyst development. However, the proportion of C1-grade embryos of the total blastocysts was significantly (P < 0.05) improved. This study suggests that addition of fructose to the culture medium can promote embryo quality and yields that are suitable for embryo transfer.

Table 1.Effect of addition of fructose on in vitro development of bovine sexed embryos
207 DEVELOPMENT OF A ROUTINE IN VITRO EMBRYO PRODUCTION SYSTEM USING SINGLY IN VITRO-MATURED, FERTILIZED, AND CULTURED BOVINE OOCYTES
Authors: Goovaerts I. G. F. , Leroy J. L. M. R. , De Clercq J. B. P. , Andries S. , Bols P. E. J.
Published: December 2008

An in vitro embryo production system (IVP), in which a single oocyte can be tracked from the moment of retrieval up to the blastocyst stage, would be a valuable tool for studies linking developmental competence and embryo metabolism to oocyte quality and follicular environment. Unfortunately, to date, data on individual IVP are inconsistent, and in most cases show unsatisfactory blastocyst rates. Earlier studies revealed that individual culture on a cumulus cell (CC) monolayer resulted in comparable numbers of good-quality embryos as obtained after regular group culture (Goovaerts et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 190). Because, in the latter study, single culture was performed after group maturation and fertilization, the aim of this study was to develop and test an individual IVP system using bovine oocytes or zygotes obtained after single maturation and single fertilization. Therefore, 532 grade I COC from slaughterhouse ovaries (3 replicates) were randomly assigned to 1 of 2 treatments: a complete individual IVP protocol, or a routine group IVP as a control. Individual maturation (TCM-199 + 20% serum) and fertilization were performed in 20-μL droplets under oil in 24-well plates. Subsequently, each zygote was cultured in 20 μL of medium (SOF + 5% serum, 90% N2, 5% CO2, 5% O2) on a 6-day-old monolayer of matured CC (5% CO2 in air). Group maturation and fertilization were carried out per 100 COC in 500 μL, whereas group culture was performed per 25 zygotes in 50-μL droplets under oil. Cleavage, blastocyst, and hatching rates were assessed 2, 8, and 10 days postfertilization, respectively. Possible effects of individual and group culture were evaluated with binary logistic regression (SPSS 15.0). No interactions between replicate and treatment could be found (P > 0.05). Cleavage and blastocyst rates were significantly lower after individual IVP, compared with group IVP, whereas the blastocyst rates on cleaved zygotes and the hatching rates did not differ significantly (Table 1). In conclusion, acceptable blastocyst rates (25.1%) could be obtained after individual IVP. The lower blastocyst rates were associated with the lower cleavage rates, and no effect of the individual embryo culture system on embryo development could be found.

Table 1.Cleavage, blastocyst, and hatching rates after individual and group in vitro embryo production (IVP)
211 IN VITRO PRODUCTION OF SWAMP BUFFALO EMBRYOS BY INTRACYTOPLASMIC SPERM INJECTION: EFFECT OF CHEMICAL ACTIVATION TREATMENTS
Authors: Liang Y. Y. , Ye D. N. , Laowtammathron C. , Phermthai T. , Parnpai R.
Published: December 2008

Intracytoplasmic spern injection (ICSI) in the buffalo has not yet been well examined. Several factors involved affect the success rates of this technique, particularly the postinjection activation procedure. The objective of this study was to evaluate the effects of chemical activation treatments on in vitro development of oocytes after ICSI. A single spermatozoa was injected into the cytoplasm of an in vitro-matured oocyte using a micromanipulator under an inverted microscope. The ICSI oocytes were assigned to the following chemical activation treatments: (1) exposed to 5 μm ionomycin (Io) in Emcare medium for 5 min and placed in Emcare medium for 3 h, or (2) exposed to 7% ethanol (EtOH) in Emcare medium for 5 min and placed in Emcare medium for 3 h. The treated oocytes that extruded a second polar body were then selected and cultured either in (A) 1.9 mm 6-dimethylaminopurine (6-DMAP) in mSOF medium for 3 h, or (B) 10 μg mL–1 of cychloheximide (CHX) for 5 h. The treated oocytes were further cultured in mSOF medium supplemented with 3 mg mL–1 of fatty acid-free BSA at 38.5°C under a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 2 d. Thereafter, 8-cell-stage embryos were selected and co-cultured with buffalo cumulus cells in mSOF medium at 38.5°C under a humidified atmosphere of 5% CO2 in air for another 5 d. The medium was changed daily and the development of embryos was recorded at the same time the medium was changed. The sham-injected oocytes were treated and cultured along with ICSI oocytes. With 8 replications for each activation treatment, 336 oocytes were used for ICSI. With 6 replications for each activation treatment, 211 oocytes were used for sham injection. The cleavage of ICSI oocytes treated with Io + 6-DMAP, EtOH + 6-DMAP, and EtOH + CHX was 76.2, 69.4, and 78.3%, respectively, which was significant higher (P < 0.01) than ICSI oocytes treated with Io + CHX (52.4%) and also significant higher (P < 0.01) than sham-injected oocytes in all treatments. The highest blastocyst rate was observed in ICSI oocytes treated with Io + 6-DMAP (28.6%), which was not significantly different from ICSI oocytes treated with EtOH + CHX (24.4%). The blastocyst rates of ICSI oocytes treated with Io + 6-DMAP and EtOH + CHX were significantly higher than ICSI oocytes treated with Io + CHX (5.9%) and EtOH + 6-DMAP (16.5%) and also were significantly higher than sham-injected oocytes in all treatments. In conclusion, our study demonstrated that activated ICSI of swamp buffalo oocytes with Io + 6-DMAP or EtOH + CHX gave the highest cleavage and blastocyst rates.

This work was supported by the Thailand Research Fund and Suranaree University of Technology.
224 USE OF STATISTICAL MODELS BASED ON BAYESIAN INFERENCE TO ESTIMATE FIELD FERTILITY OF NELORE BULLS BY USING DATA OBTAINED IN AN IN VITRO FERTILIZATION PROGRAM
Authors: Sudano M. J. , Crespilho A. M. , Martins A. , Papa F. O. , Rodrigues J. , Landim-Alvarenga F. C.
Published: December 2008

The aim of the present study was to analyze the use of in vitro fertilization to predict the field fertility of Nelore bulls by using statistical models based on Bayesian inference. A total of 407 oocytes were obtained from slaughterhouse ovaries and submitted to in vitro maturation in TCM-199 and fertilization in Tyrode’s + BSA + lactate + pyruvate. Sperm cells from 3 different bulls, namely, V, T, and G, were used. All bulls had a known field gestational rate. After 18 h of IVF, a sample of zygotes (n = 40/bull) was stripped and stained with acetic orcein (1%) to estimate the number of pronuclei. The remaining zygotes (n = 287) were cultured in SOF for 7 to 8 days. The results are based on 3 replicates with 32 to 33 oocytes/bull per replicate. All 3 bulls analyzed presented similar percentages of oocytes with at least 2 pronuclei (52.5, 40, and 40% for bulls V, T, and G, respectively). However, when cleavage rate was analyzed, bull T presented a significantly (P = 0.0022) lower rate (57.4%) compared with bulls V (77.9%) and G (78.1%). In contrast, blastocyst formation rates were similar between bulls (27.9, 22.8, and 30.5% for bulls V, T, and G, respectively). When the cleavage and blastocyst rates were analyzed in comparison with the field pregnancy rates previously obtained for bulls V, T, and G by using statistic models based on Bayesian inference, the predictions of conception rate based on cleavage were 59.8 ± 2.5%, 54.2 ± 4.7%, and 59.8 ± 2.5% for bulls V, T, and G, respectively. When blastocyst formation rates were used, the predicted conception rate was 58.5 ± 2.2%, 52.3 ± 4.5%, and 61.5 ± 2.8% for bulls V, T, and G, respectively. The predicted conception rates obtained by using the cleavage and blastocyst data together were 54.7 ± 3.9%, 54.1 ± 4.8%, and 63.0 ± 3.1% for bulls V, T, and G, respectively, which was very similar to the real field conception rates for the same bulls (54.4, 54.1, and 63.3%, respectively). The statistical model obtained through the use of Bayesian inference with the combined data for cleavage and blastocyst rates presented a good correlation with the real field fertility of bulls used in this experiment. This result indicates that this statistical method could be a good and efficient alternative to predict the fertility of Nelore bulls. However, a larger number of animals will need to be tested by the same methodology to confirm these results.

FAPESP.
225 ANTI-APOPTOTIC EFFECT OF AGGREGATION ON PREIMPLANTATION DEVELOPMENT OF BOVINE IN VITRO-FERTILIZED EMBRYOS
Authors: Yoon S. W. , Park C. H. , Lee S. G. , Kim H. M. , Park J. K. , Uh K. J. , Kim H. S. , Lee C. K.
Published: December 2008

Apoptosis occurs during embryonic development, and is related to early embryonic loss. It is important to produce high-quality blastocysts in vitro for research on the establishment of embryonic stem (ES) cells and transgenic animal production. Therefore, our objectives were to compare the anti-apoptotic effect of bovine aggregate v. nonaggregate IVF embryos and to determine whether aggregation could improve the quality of bovine embryos. The cumulus–oocyte complexes were matured for 20–22 h, and the oocytes were fertilized with cryo-preserved bovine sperm using the swim-up method. After removal of the zona pellucida (ZP), three 4-cell-stage embryos (3X) were aggregated by co-culture in an aggregation hole that was made by an aggregation needle on the culture dish. Embryos were cultured either singularly (1X, ZP removed) or in aggregates of three (3X), and IVF intact embryos served as a control. Five days after aggregation, the developmental rate was observed. The numbers of total cells and apoptotic cells were determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay using blastocyst-stage embryos. Moreover, the mRNA expression pattern related to apoptosis and embryo quality was verified by real-time PCR of the aggregated (3X) and nonaggregated (1X) embryos (at least 3 embryos). The percentage of blastocysts was higher in the 3X aggregated embryos (41.3%) compared with that of the 1X ZP-free embryos (24.3%), whereas there was no significant difference in the 1X embryos and the intact controls (24.3 and 25.8%, respectively; P < 0.05). The total cell number of blastocysts also increased approximately threefold (P < 0.05) in 3X aggregated embryos compared with that of 1X controls. In contrast, the percentage of TUNEL-positive cells, an indication of apoptotic cells, was decreased by approximately threefold in 3X aggregated embryos when compared with that of 1X embryos (7.7 and 2.6%, respectively). The mRNA levels for the Oct-4, NANOG, and bcl-2 genes were higher (P < 0.05) and for the Bax gene were lower in the 3X aggregated embryos than for those of the 1X controls. Therefore, our results indicated that aggregation of bovine IVF embryos at a 4-cell stage could promote the quality and suppress the apoptosis of bovine pre-implantation-stage embryos produced in vitro. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the establishment rate of ES cell lines by seeding on the feeder layer and raising the efficiency of embryo transfer.

This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).
228 BOAR FERTILITY AND SPERM CHROMATIN STRUCTURE ASSAY DEFINED SPERM DNA FRAGMENTATION
Authors: Evenson D. P. , Kasperson K. , Wixon R. L. , Didion B. A.
Published: December 2008

The sperm chromatin structure assay (SCSA) was used retrospectively to characterize sperm from 18 sexually mature boars having fertility information. Boar fertility was defined by farrowing rate (FR) and average total number of pigs born (ANB) per litter of gilts and sows mated to individual boars. The SCSA uses flow cytometry to evaluate the structural integrity of sperm nuclear DNA. In brief, aliquots of frozen/thawed semen were treated for 30 s with a pH 1.2 buffer that denatures sperm DNA at the sites of DNA strand breaks. The samples are then stained with acridine orange (AO) that intercalates into native, double-stranded DNA and fluoresces green under the flow cytometer laser beam. Broken and denatured single-stranded DNA associates with AO and then collapses, causing a metachromatic shift from green to red fluorescence. Five thousand sperm are measured per sample at rates of ~200 s–1, thus giving the data strong statistical robustness. This fact coupled with the precision of the AO/DNA biochemistry provides a CV ~2% between replicate samples. The SCSA parameters measured in this study were the %DNA fragmentation index (%DFI; percentage of sperm with fragmented DNA) and standard deviation of DFI (SD DFI). The %DFI and SD DFI showed the following significant negative correlations with FR and ANB; %DFI v. FR r = –0.55, P < 0.01, SD DFI v. FR r = –0.67, P < 0.002, %DFI v. ANB r = –0.54, P < 0.01, and SD DFI v. ANB r = –0.54, P < 0.02. A discrimination analysis was used to distinguish between the boar groups above and below 6% DFI. The variables most useful to discriminate between 2 groups were SD DFI, FR, and ANB. The average squared canonical correlation for SD DFI was 0.82, P < 0.0001. The average squared canonical correlation for FR and ANB was 0.45, P < 0.003 and 0.54, P < 0.001, respectively. Odds ratios for %DFI and SD DFI were 1.5 times (P = 0.0003, CI 1.22, 1.94) and 2.5 times (P = 0.0001, CI 1.87, 3.32), respectively.
252 EFFECTS OF IN VITRO MATURATION AND AGE ON OOCYTE METAPHASE SPINDLE INTEGRITY AND CHROMOSOME SEGREGATION IN THE RHESUS MONKEY
Authors: Nichols S. M. , Gonzalez-Martinez J. A. , Bavister B. D.
Published: December 2008

Oocyte IVM offers tremendous potential for the treatment of infertility by reducing the costs, risks, and side effects associated with ovarian stimulation. Although normal offspring have been produced using IVM/IVF technology in humans and monkeys, IVM is far from being a reliable technique for the production of developmentally competent oocytes in primates. This study examined oocyte karyotype and spindle integrity in rhesus monkey oocytes, and attempted to correlate anomalies with IVM and age of oocyte donors. Each of 14 females (ages 6 to 22 years) underwent both regimen A (FSH + hCG) and regimen B (FSH only) stimulation cycles at least once over the study period to facilitate collection of both mature and immature oocytes. Immature oocytes from regimens A and B underwent IVM to produce metaphase II oocytes. Metaphase II oocytes from each collection cycle cohort were fixed for either karyotype or metaphase spindle analysis. Statistical analysis of aneuploidy rates and spindle aberrations was performed using 2 × 2 G tests. Karyotype analysis revealed a significantly lower rate of aneuploidy in in vivo-matured (IVO) oocytes (6.0%) compared with IVM oocytes from regimen B (19.8%). Factoring age into these analyses revealed a significant difference in aneuploidy rate between IVO oocytes from young females (4.7%) and regimen B IVM oocytes from old females (25.0%; P < 0.05). Confocal analysis demonstrated a significant increase in metaphase spindle anomalies in IVM oocytes compared with IVO oocytes, which could be attributed to a significant increase in the rate of abnormal chromosome alignment on the metaphase spindle in IVM oocytes originating from either stimulation regimen: 7.0% in IVO oocytes v. 27.9% (regimen A) and 32.6% (regimen B) in IVM oocytes (P < 0.05). When donor female age was considered, regimen B IVM oocytes from old females displayed abnormal chromosome alignment on the spindle equator at a significantly higher rate than oocytes of any other maturation condition or age group (P < 0.05). In addition, IVO oocytes from young females showed this anomaly at a significantly lower rate than regimen B IVM oocytes from the same young females. We conclude that IVM can induce meiotic anomalies in the macaque oocyte, especially those obtained from older females. The results from this study provide possible explanations for the reported reduction in developmental competence of IVM v. IVO primate oocytes. This study provides information on the nuclear aspects of IVM, namely, the effect of maturation conditions and age on incidences of abnormal chromosome segregation during meiosis I and abnormal chromosome congression during meiosis II, data that may be helpful for determining optimal IVM culture conditions.
256 LEPTIN AND GLUCOSE INFLUENCE PORCINE NUCLEAR MATURATION
Authors: Silva E. , Krisher R. L.
Published: December 2008

Leptin (LEP), a product of the obese gene, regulates food intake and may be associated with the metabolic syndrome. In addition, leptin receptor has been identified in the luteal and granulosa cells of the pig, suggesting that this protein may play a role in fertility. Research indicates that LEP is associated with insulin sensitivity; thus, the addition of different concentrations of LEP and glucose (GLUC) in maturation media may have an impact on oocyte maturation. The objective of the current experiments was to determine the effect of LEP (Experiment 1), and LEP in combination with GLUC (Experiment 2) on the nuclear maturation of porcine oocytes. This could potentially be a good model system in which to study the effects of obesity and diabetes on oocyte quality. Cumulus–oocyte complexes were cultured in a chemically defined medium, Purdue porcine medium (PPM) for 42 h, in 7% CO2 in air and at 38.7°C. In Experiment 1, medium was supplemented with 5 different concentrations (0, 1, 10, 20, and 100 ng mL–1) of LEP, in the presence of 2 mm GLUC. Experiment 2 was designed as a 3 × 3 factorial, with LEP (0, 1, and 10 ng mL–1) and GLUC (0, 5, and 50 mm). Oocytes were fixed and stained after IVM (136 to 155/treatment, 7 replicates; and 39 to 90/treatment, 5 replicates for Experiment 1 and 2, respectively). Nuclear maturation was scored as 1 (mature; T or MII) or 0 (not mature). Data were analyzed by GLM ANOVA and chi-square. There was no difference in nuclear maturation between 0, 1, 10, 20, and 100 ng mL–1 LEP (79.4, 82.3, 74.5, 77.2, and 83.7% mature, respectively). According to these data, LEP alone did not have an effect on in vitro maturation of porcine oocytes when using a chemically defined maturation medium. Absence of GLUC in the medium had a negative effect on maturation (39.5%; P < 0.01) compared with treatment with 5 mm (88.2%) and 50 mm (74.7%) GLUC, independent of LEP. In the presence of 5 mm GLUC, there was no difference between 0, 1, and 10 ng mL–1 LEP (83.9, 94.9, and 88.3%, respectively). However, LEP did affect nuclear maturation when COC were cultured in the absence of GLUC or with excessive GLUC. Leptin (10 ng mL–1) tended (P = 0.1) to promote nuclear maturation (46.2%) in the absence of GLUC (0 mm), compared with 0 ng mL–1 LEP (32.3%). In high-GLUC (50 mm) medium, 1 ng mL–1 LEP had a positive effect (P < 0.05) on nuclear maturation (87.8%) compared with 0 ng mL–1 (66.7%) and 10 ng mL–1 LEP (72.3%). LEP (0 or 10 ng mL–1) inhibited (P < 0.05) nuclear maturation in the presence of high (50 mm) GLUC compared with that in 5 mm GLUC. These data demonstrate that addition of 10 ng mL–1 LEP in maturation media may mitigate the negative effect of maturation in the absence of GLUC. Moreover, in the presence of excessive GLUC, 1 ng mL–1 LEP stimulates nuclear maturation. These results suggest that LEP and GLUC may interact to regulate oocyte nuclear maturation in obese or diabetic individuals, or both.