bluuebunny/crossref_metadata_2025 huggingface dataset:
10 TRACEABILITY SYSTEM FOR AN INDIVIDUAL FROZEN SEMEN STRAW BY A TINY RADIO FREQUENCY IDENTIFICATION CHIP
Authors: Hoshino Y. , Mukojima K. , Minami N. , Imai H.
Published: December 2011

A traceability of frozen semen straws that certify the bloodline of a sire will improve the efficiency of cattle breeding. Semen of the sire can be generally identified by the colour of a straw and printing on a straw. However, it is difficult to identify individual frozen semen straws by the conventional method. To identify straws individually, each straw must be tagged with a unique ID that is able to be read quickly in the frozen state. We have established the identification method for individual frozen semen straws using radio frequency identification (RFID) and developed a traceability system that is able to record the distribution history of an individual semen straw from the production of frozen semen to artificial insemination (AI). We used a 2.5-mm diameter straw combined with an RFID tag, which consisted of a tiny RFID chip (μ-Chip: Hitachi Co. Ltd, 0.4 × 0.4 × 0.2 mm) and a thin aluminum antenna on the polyester sheet (54 × 1.5 × 0.05 mm). The μ-chip contains a unique ID (128 bit). It responds to the 2.45 GHz microwave frequency that is emitted from the RFID reader and transmits its ID to the RFID reader. The μ-Chip retains its function on a straw, which has been stored in the liquid nitrogen (–196°C). Artificial insemination of the frozen semen straw with the RFID tag could be performed using a conventional AI gun. The semen traceability system consisted of the database server and the client software. Information about semen straws is connected with their ID and stored on the database server. Producers and distributors of frozen semen can count the number of semen straws correctly by reading their ID one by one and can send information to the database server through the web by easy operation using the client software. Artificial insemination information can be instantly recorded by the handy terminal that is able to read both a bar-code on the ear tag of the cow and the RFID tag of the straw. The system has been used on fields at Gifu Prefecture in Japan for ∼3 months. Eight hundred thirty-one frozen semen straws with the RFID tag were produced from 3 bulls. After freezing of the straws, 17 straws were not identified with their ID. It was speculated that their μ-Chips were disconnected from the antenna during freezing. Six hundred ninety-four straws were distributed to the 7 AI centers. Five hundred thirty straws were used for AI by 15 inseminators to a total of 424 cows at the 101 farms. Although only one straw could not read its ID after AI, information of the remaining 529 straws were recorded on the database accurately. A strict traceability of frozen semen straws can be established using the individual identification of straws by RFID. Information stored in the database would be useful for the evaluation of sire and the reproductive management of cows.
This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (MAFF).
103 OVIDUCTAL SECRETORY PROTEINS AS MEDIA SUPPLEMENT FOR IN VITRO EMBRYO DEVELOPMENT IN CATTLE
Authors: Das S. K. , Sharma A. K. , Bhatia V. , Mohanty A. K.
Published: December 2011

The objective of this study was to determine the effect of oviducal secretory proteins (cOSP) as a media supplement on in vitro embryo development in cattle. Oviducal secretory proteins were collected from slaughterhouse oviducts by repeated freeze–thaw process and purified by ammonium sulfate precipitation (30%, 40%, 50% and 60%) followed by dialysis in 50 mM tris-HCl (pH 7.0) buffer. Dialyzed products were further purified by SP Sephadex cation exchange column and diethylaminoethyl cellulose (DEAE) anion exchange column, extensively washed and eluted by 50 mM tris-HCl (pH 7.0) containing 1.5 M NaCl. Both bound and unbound proteins were collected separately, dialyzed in phosphate buffer saline and quantified. Presence of protein was confirmed by running sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and OSP bands between ∼66 kD to ∼97 kD were found. The pooled purified cOSP were used as a media supplement in 3 different concentrations (0, 10, 50 and 100 μg mL–1) for in vitro production (n = 3) of cattle embryos. Cumulus–oocyte complexes (n = 370) were collected from slaughterhouse ovaries, washed thoroughly and cultured in maturation media for 24 h in 5% CO2 at 38.5°C with maximum humidity. In vitro-matured oocytes were fertilized with in vitro capacitated sperm in Fert-BO media at 38.5°C in 5% CO2. After 16 to 18 h, oocytes were washed and cultured in embryo development media for cleavage. After 40 to 42 h, cleavage was observed and embryos were transferred into the replacement media for further development. A total of 68.42%, 69.31%, 61.82% and 41.67% cleavage rate and 15.38%, 21.31%, 14.70% and 15.0% blastocyst rate was observed at concentrations of 0, 10, 50 and 100 μg mL–1, respectively. These results indicate that addition of cOSP at 10 μg mL–1 increased blastocyst formation significantly (P < 0.05) compared with 0, 50 and 100 μg mL–1 and increased cleavage rate significantly (P < 0.05) compared with 50 and 100 μg mL–1.
The authors acknowledge sincere thanks to the Director, Joint Director (Research), National Dairy Research Institute, Karnal and Incharge, Eastern Regional Station, National Dairy Research Institute, Kalyani, for providing the necessary facilities to carry out the work.
107 UNEXPLAINED VARIATION IN EMBRYO FREEZER DIGITAL READOUT
Authors: Nelson C. F. , Nelson L. D.
Published: December 2011

Differences among frozen embryo transfer results are often observed but are usually unexplainable. One cause of possible variability in pregnancy rates could be faulty digital readout of the biological freezer unless adequate controls are routinely used. By monitoring temperatures throughout the freezing process independently of the freezer readout, differences between the embryo freezer digital readout (EFDR) vs thermocouple (TC) and digital thermometer (DT) were observed when using a Bio Cool II Alcohol Freezer. The difference between EFDR and actual temperature (TC and DT) ranged from –4.9°C to +5.4°C. To further investigate possible causes of these differences, electrical supply to the freezer and ambient temperatures were monitored at 21 different on-the-farm locations. Ranges of voltage (117–124.4), Hz (59.96–60.00), mA (10.82–11.90) and ambient temperature (–6.1°C to +23.9°C) were recorded. However, no correlations between the EFDR and voltage (r = 0.097), Hz (r = –0.227), mA (r = 0.140), or actual temperature (r = –0.350) were found (P > 0.05). Two freezing procedures were done in unheated barn areas where the ambient temperature was –6.1°C and –4.9°C, with readout differences of +1.7°C and –0.8°C, respectively. Six of the 21 differences were ≥+2.0°C and 4 differences were ≤–2.0°C. The variable EFDR was apparently a problem within the freezer itself. In practice the real temperature (TC and DT) was used to determine the seeding temperature, therefore eliminating the chance of seeding at highly variable temperatures (–10°C to –2°C). The readout difference remained consistent throughout the freezing process and did not affect the freezing curve. It is suggested that any laboratory instrument be independently monitored to ensure quality control.
110 PREGNANCY RATES OF BOVINE EMBRYOS AND HEMI-EMBRYOS TRANSFERENCE
Authors: Torres C. A. A. , Fernandes C. A. C. , Oliveira F. A. , Penitente Filho J. M. , Oliveira C. T. S. A. M. , Santos M. C. R. , Jiménez C. R. , Triana E. L. C. , Oliveira M. M. N. F.
Published: December 2011

Embryo transfer (ET) in cattle speeds up genetic gains, but its use is limited by cost-benefit analysis. In addition other techniques can be developed, such as sexing and bipartition of embryos. An alternative to improve the economic viability of ET in cattle would be to bipartition embryos in order to increase the yield of the technique. The aim of this study was to investigate the operational viability of embryo bipartition technique in an ET program in cattle and to study aspects related to the viability and development of bovine hemi-embryos (HE) compared with intact ones. The embryos were collected by nonsurgical technique 7 days after the onset of oestrus. Viable structures (130) from 49 embryo collections from 25 Aberdeen and Simmental donor cows and heifers were used. Only embryos in compact morula, early blastocyst and blastocyst stage, with a morphological range from excellent to good grade (IETS Grade 1), were split. The embryos, split (78) or not (52), were transferred into the uterine horn ipsilateral to the corpus luteum of the recipients 7 days after oestrus. The treatment groups evaluated were T1 (intact embryo, n = 52), T2 (1 HE, n = 27) and T3 (2 HE, n = 51). Crossbred heifers were used as recipients and pregnancy diagnosis was done at 60 to 80 days of gestation. The embryos of T1, T2 and T3 were classified morphologically as excellent or good and by developmental stage as morula, early blastocyst, or blastocyst, distributed as follows: T1: 30, 22 and 16, 17 and 19; T2: 15, 12 and 7, 9 and 11; and T3: 29, 22 and 15, 15 and 21, respectively. The birth rate per original embryos was greater for T2 than for T1 and T3 (Table 1). The pregnancy rates for excellent and good embryos and morulae, early blastocysts and blastocysts were not different (P > 0.05). The T1, T2 and T3 twin births were 0, 1 and 5, respectively. It is concluded that embryo bipartition technique applied in a commercial ET program is a viable operational technique.

Table 1.Pregnancy at 60 to 80 days and birth rate per original embryos used

Supported by CAPES, CNPq, FAPEMIG and BIOTRAN.
113 INTERACTIONS BETWEEN THE VISCERAL YOLK SAC PLACENTA AND THE UTERINE WALL IN NECROMYS LASIURUS (RODENTIA, CRICETIDAE)—AN EXPERIMENTAL MODEL TO UNDERSTANDING THE MATERNAL–FETAL EXCHANGE
Authors: Favaron P. O. , Carter A. M. , Mess A. M. , Oliveira M. F. , Miglino M. A.
Published: December 2011

The establishment of pregnancy results from the interaction between the trophoblast and maternal tissues. Evolutionarily, the yolk sac (YS) is the only fetal membrane present in all vertebrate taxa. In mammals, it results in the development of the YS placenta, which is responsible for maternal–fetal exchange in the beginning of gestation. Its functions are related to hematopoiesis, selective transfer of various substances including proteins, iron transfer and histiotrophic nutrition. Necromys lasiurus is a rodent species from South America, which is related to species which are used as laboratory animals. Herein, we show data on the early development of the YS to promote a better understanding of its relationship with the uterine wall. Ten implantation sites of Necromys were obtained from a breeding group at the UFERSA, Mossoró, Brazil. Samples were examined by means of histology, immunohistochemistry for vimentin (1:200) and Proliferating Cell Nuclear Antigen (PCNA; 1:800) and scanning and transmission electron microscopy. In early pregnancy the visceral YS was in places closely attached to the uterine wall. At these attachments, the uterus possessed infolded structures with a high-columnar epithelium associated with blood vessels and surrounded by vimentin positive glands. In these areas, the YS became compact in shape and we observed an intimate association of both visceral yolk sac and endometrial luminal epithelium. A strong response to periodic acid Schiff, including plenty of granular vesicles, indicated secretory activity of the endodermal cells, such as release of histiotrophe. Large intercellular spaces, vacuoles and glycogen were present in the cytoplasm of these cells. On the other hand, in areas where the visceral YS was near to the labyrinthine region of chorioallantoic placenta, it was highly villous. The villi were formed by endoderm cells with hexagonal shape. The visceral YS was supplied by fetal vessels, which form the blood islands, structures related to the fetal hematopoiesis. A prominent microvilli area was observed on the surface of the endoderm cells. Additionally, vacuoles, electron-dense inclusions and dense droplets indicative of high secretory and transfer activities were seen inside of these cells. According to PCNA labelling, the mesothelial layer in the region near to uterine consisted of proliferative cells. Although early gestation represents a crucial developmental stage, information about the interaction between the trophoblast and maternal tissues is sparse. Embryonic mortality, a major cause of reproductive failure in livestock species, can be related to interactive problems between maternal and fetal tissues that affect exchange of nutrients, hematopoiesis and other vital functions.
Supported by grants from FAPESP (Proc. 09/53392-8).
115 FIRST SUCCESSFUL PREGNANCY AFTER EMBRYO TRANSFER INTO SYNCHRONIZED FEMALES IN GUINEA PIG (CAVIA PORCELLUS)
Authors: Grégoire A. , Peredo F. , León S. , Huamán E. , Allard A. , Joly T.
Published: December 2011

The guinea pig (Cavia porcellus) has been used as a laboratory animal since the late 18th century and still remains essential in many research areas. It also plays an important role in the Andes societies as a source of protein for many low-income highlanders and as part of rituals and traditional medicines. Thus, the conservation of genetic diversity is a long-term issue that must be considered. To establish an embryo cryobank, it is necessary to develop a method of embryo transfer. Up to now no pregnancies after surgical embryo transfer into synchronized females have been reported in guinea pigs. The aim of this work was to design a standard embryo transfer method in this species. Eight normally cycling female guinea pigs from the Maria-Marcela Farm (Puente Piedra, Peru), weighing from 1 to 1.5 kg, were used in this study. Females were housed under farming conditions and fed on commercial pellets and tap water ad libitum. Three donor females were superovulated using 15 IU of human menopausal gonadotrophin (hMG, Massone®, Buenos Aires, Argentina) and mated as soon as the vagina opened. Copulatory plug was observed and vaginal smears were taken to guarantee successful mating. Thirty-eight embryos were collected between Days 3.5 and 4.5 after ovulation at the morula and early blastocyst stages. Five recipient females were synchronized by a daily 0.1-mL dose of altrenogest (Regumate® Equine, Intervet, France) per os by means of a syringe for 15 days. Two embryos were transferred into each uterine horn by laparotomy at Day 3.5 and 4.5 after ovulation. Two types of pipettes were tested for embryo transfer: pulled glass pipettes approximately 0.3 mm in diameter in 2 female recipients and plastic open pulled straws (OPS, Minitüb®, Germany) in 3 recipients. Pregnancy diagnosis was detected by observation of no return to oestrus at Day 16 and confirmed by ultrasonography. None of the 3 OPS-transferred females were pregnant. One of the 2 pulled glass pipette–transferred females was diagnosed as pregnant and delivered 2 stillbirths (one per uterine horn). There were no postsurgical complications and the females undergoing embryo transfer returned to normal reproduction. We demonstrated that a classic surgical embryo transfer method is possible under field conditions to obtain pregnancy in this species. We suggest further studies using glass pipettes, which allow a more precise embryo deposition. Future experiments will incorporate the transfer of frozen-thawed embryos on a larger scale.
118 INDUCING PLURIPOTENCY IN SOMATIC CELLS FROM THE SNOW LEOPARD (PANTHERA UNCIA), AN ENDANGERED FELID
Authors: Verma R. , Holland M. , Smith P. , Verma P.
Published: December 2011

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMX) encoding either 4 (Oct-4, Sox-2, Klf-4 and cMyc) or 5 (Oct-4, Sox-2, Klf-4, cMyc and Nanog) human transcription factors. Transduction efficiency of the retrovirus was ascertained using pMX-green fluorescent protein transgene expression and averaged 96% from 3 repeated experiments. The reprogramming efficiency of initial colony formation was 0.000308% (37/120 000 cells plated) for 4-factor induction compared with 0.000517% (62/120 000) for 5-factor induction. Transduction with 4 factors resulted in the formation of small colonies of cells, which could not be maintained for more than 4 passages (P4). However, addition of Nanog to the transfection cocktail produced stable iPS cell colonies, which formed as early as Day 3. Colonies of cells were selected at Day 5 and expanded in vitro on mouse embryonic fibroblast feeder cells. The resulting cell line was positive for alkaline phosphatase, Oct-4, Nanog and stage-specific embryonic antigen-4 at P14. Also, RT-PCR confirmed that endogenous Oct-4 and Nanog were expressed by snow leopard iPS cells from P4; although all 5 human transgenes were transcribed at P4, Oct-4, Sox-2 and Nanog transgenes were silenced as early as P14, suggesting that reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the 3 germ layers. This study describes the first derivation of iPS cells from the endangered snow leopard and is also the first report on induced pluripotency in felid species. Our results demonstrate that addition of Nanog to the reprogramming cocktail was essential for derivation of iPS lines in this felid and that iPS cells provide a unique source of pluripotent cells with utility in conservation for cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.
121 ALGINATE-FIBRIN GEL MATRIX PROMOTES IN VITRO GROWTH OF DOG SECONDARY FOLLICLES
Authors: Songsasen N. , Guzy C. , Wildt D. E.
Published: December 2011

A previous study from our laboratory has demonstrated that preantral follicles from the dog that are cultured in alginate are able to grow and produce steroid hormones (Songsasen et al. 2011 Reproduction 142, 113–122). Here we investigated the influence of using a combination of alginate and a degradable biomaterial, fibrin, on dog follicle development in vitro. We hypothesised that the alginate and fibrin gel matrix would be superior to alginate alone because the former has dynamic mechanical properties that permit more expansive follicle development than the inert alginate-only system. Secondary follicles (128–220 μm in diameter) were collected from the ovaries of 4 prepubertal dogs (<6 months of age) and encapsulated in 0.5% alginate (n = 26) or 0.5% alginate + 12.5 mg mL–1 of fibrin (n = 22). Follicles were cultured for 12 days at 38.5°C in 100 μL of α-minimal essential medium + 2 mM of glutamine + 5.5 μg mL–1 of insulin + 5.5 μg mL–1 of transferrin + 6.7 ng mL–1 of selenium + 10 μg mL–1 of FSH and 1 ng mL–1 of LH + 3 mg mL–1 of polyvinyl alcohol. Follicle diameter was monitored and half of the medium exchanged every 48 h. Follicle survival was assessed based on ability to increase in size, as well as on oocyte and granulosa cell morphology. Comparisons of follicle growth rate for each culture day between the 2 treatments were conducted using Student's t-test and among culture days within the same group using ANOVA followed by a Holm-Sidak multiple comparison. Follicle survival was compared using a chi-square test. In both groups, follicles maintained the 3-dimensional structure and increased (P < 0.05) in size as culture period progressed. However, follicles encapsulated in alginate + fibrin grew larger (P < 0.05) than those in alginate alone. Specifically, follicles in alginate + fibrin were doubled in size by 12 days compared with a 60% increase for alginate alone. There were no differences (P > 0.05) in follicle survival between the 2 groups (27.0 and 38.1% for alginate and alginate-fibrin, respectively). Results demonstrate that a dynamic alginate-fibrin matrix enhances in vitro follicle growth. We suspect that the mechanism involved is related to facilitating expansion capacity. Specifically, it is likely that nondegradable alginate offers physical, but eventually restrictive, support to encapsulated cells. By contrast, in the gel combination, the fibrin degrades due to cell-secreted proteases that, in turn, permit more robust follicle expansion. Low follicle survival (<40%) in both treatments emphasises the need for more studies to identify influential endocrine/paracrine factors that enhance follicle growth and production of competent oocytes.
Funded by NIH-KO1RR020564.
129 EFFECT OF INSULIN-LIKE GROWTH FACTOR 1 AND FOLLICLE-STIMULATING HORMONE IN A DYNAMIC MEDIUM ON VIABILITY AND FOLLICULAR DEVELOPMENT OF CAPRINE EARLY PREANTRAL FOLLICLES
Authors: Magalhães-Padilha D. , Gabriela F. , Haag K. , Gastal M. , Jones K. , Figueiredo J. R. , Gastal E.
Published: December 2011

The goal of this study was to investigate the effect of a dynamic medium supplemented with insulin-like growth factor 1 (IGF-1), FSH, or both on the viability, activation and secondary follicle rates and on the follicle and oocyte diameters of caprine preantral follicles submitted to a long-term (16 days of culture) in vitro culture system. Fragments from goat ovaries (n = 16) obtained from slaughterhouse were cultured in α-MEM containing or not containing IGF-1 (50 ng mL–1), FSH (50 ng mL–1), or both as a dynamic medium that was added in the first (Day 0 to 8) and second (Day 8 to 16) halves of culture, resulting in 6 treatments: α-MEM alone, IGF-1/IGF-1, FSH/FSH, IGF-1/FSH, FSH/IGF-1 and IGF-1 + FSH/IGF-1 + FSH. Noncultured (fresh control) and cultured fragments were processed for morphological and viability analyses. Follicles within ovarian fragments were mechanically isolated and early-stage follicles were classified as normal or abnormal and primordial, primary, or secondary. The viability of isolated follicles was determined by trypan blue dye and follicles were classified as live or dead. For this experiment, 6240 preantral follicles were analysed. Data for statistical analyses were transformed and submitted to ANOVA using the GLM procedure of SAS, followed by the Duncan test for comparison of means. At Day 8 of culture, more (P < 0.05) follicles in the treatments containing IGF-1 alone or associated with FSH were normal and viable than in the treatments cultured with FSH or α-MEM alone. At Day 16 of culture, the highest (P < 0.05) percentage of viability was observed in the IGF-1/IGF-1 (68%), IGF-1/FSH (68%) and IGF-1 + FSH/IGF-1 + FSH (72%) treatments. However, more (P < 0.05) normal follicles were observed on Day 16 in the IGF-1 + FSH/IGF-1 + FSH treatment (76%) than in all other treatments, except for the IGF-1/FSH treatment (72%). The percentage of follicular activation (primordial to primary) increased (P < 0.05) in all treatments from Day 0 to 8 (mean, 5 to 49%, respectively). The rate of follicular activation increased (P < 0.05) from Day 8 to 16 in all groups, except for the FSH/FSH and IGF-1 + FSH/IGF-1 + FSH treatments. Nevertheless, at Day 16, the IGF-1 + FSH/IGF-1 + FSH treatment had the highest (P < 0.05) percentage of secondary follicles (28%) when compared with the other groups (range, 6 to 17%). Furthermore, the IGF-1 + FSH/IGF-1 + FSH treatment had the largest (P < 0.05) mean follicular and oocyte diameters after 16 days of culture. In summary, follicular morphology and viability were maintained, follicle activation was promoted and secondary follicle formation was stimulated with the association of IGF-1 and FSH. Therefore, our results demonstrate the importance of IGF-1 associated with FSH during the entire long-term culture period on early folliculogenesis in goats.
130 EXPRESSION OF STEROIDOGENIC ENZYMES IN THE CAT OVARY DURING FOLLICULAR GROWTH
Authors: Halter S. , Reynaud K. , Malandain E. , Chebrout M. , Thoumire S. , Chastant-Maillard S.
Published: December 2011

This study was designed to describe the expression of steroidogenic enzymes in the various compartments of the feline ovary, from primordial to preovulatory follicles: P450 side-chain cleavage (P450scc), 17α-hydroxylase (17α-OH), 3β-hydroxysteroid dehydrogenase (3β-HSD). Nine female cats were ovariectomized during behavioural oestrus. After validation of their specificity by Western blot on feline tissue, 3 polyclonal antibodies obtained in the rabbit were used for immunohistochemistry (anti-bovine P450scc; anti-pig 17α-OH; anti-human 3β-HSD). Three successive ovarian cryosections were incubated with antibodies and 2 sections were used as controls (purified immunoglobulin G from a nonimmunized rabbit). Immunostaining was revealed by streptavidin-biotin system (LSAB kit, DAKO S.A., Trappes, France), with aminoethylcarbazole as a chromogen. The staining was evaluated according to its localization, intensity (from 1 to 4) and number of cell layers stained. Histological examination after haematoxylin-eosin-safran staining allowed follicle diameter measurement and evaluation of the atresia. Analysis of variance was used to compare follicles from various size classes. A total of 343 follicles from 140 to 3250 μm in diameter were observed (i.e. all follicles present on the slides), of which 15.2% were atretic. The observations were similar for the 3 enzymes. From the preantral stage onward (diameter >140 μm), more than 85% of the healthy follicles had the theca interna positive for P450scc, 3β-HSD and 17α-OH (respectively, 87.3, 86.0 and 99.0%). Approximately 20 to 25% were also positive in the theca externa. Granulosa were stained in 9.5% (P450scc), 0.8% (3β-HSD) and 1.8% (17α-OH) of the healthy follicles, but always with a low intensity (<2). The follicular size did not affect the proportion of stained follicles, but the staining intensity decreased progressively until 1200 μm and increased thereafter. The number of positive layers decreased continuously until 1500 μm in diameter. Between 90 and 95% of the preovulatory follicles (>2000 μm) were positive in the theca interna; the signal was more intense and the number of stained layers was higher than in smaller follicles. The 3 enzymes were detected in all the atretic follicles, with a similar intensity as in healthy ones; they were also present in the interstitial tissue, but with a higher intensity. The staining pattern in the various ovarian compartments suggests that the major steroidogenic tissues in the cat ovary are the theca interna of healthy follicles and the interstitial tissue. The lack of expression of the 3 key enzymes in the granulosa cells, even in preovulatory follicles, strongly suggests that these cells, shaping the future corpus luteum, are probably unable to produce progesterone.
131 EFFECT OF PARABENS ON THE DISRUPTION OF OVARIAN FOLLICLE MATURATION IN A NEONATAL FEMALE RAT MODEL
Authors: Ahn H. J. , Choi K. C. , Jeung E. B.
Published: December 2011

Parabens are widely used as an antimicrobial agent in the preparation of cosmetic consumer products and pharmaceuticals. Parabens are esters of para-hydroxybenzoic acid typically including methyl-, ethyl-, butyl-, isobutyl- and isopropyl-paraben. Recently, parabens have been shown to act as xenoestrogens, a class of endocrine disruptors. In this study, we hypothesised that parabens might disrupt ovarian follicle maturation in female Sprague-Dawley rats during the neonatal period. A total of 55 female rats were divided into 11 groups, each composed of 5 rats. The pups were given daily subcutaneous injections of methyl-, propyl- or butyl-paraben at increasing concentrations of 62.5, 250, or 1000 mg kg–1 per day on neonatal Day 1 to 7. 17α-Ethinylestradiol (1 mg kg–1 per day) was used as a positive control. We examined the expression of anti-Müllerian hormone (AMH), which inhibits early folliculogenesis in the ovaries. Levels of AMH mRNA and protein were determined using real-time PCR and Western blot analysis, respectively. We found that the levels of AMH mRNA and protein were dose-dependently increased by methyl- and propyl-paraben compared with the vehicle. In particular, AMH mRNA and protein levels were markedly increased by parabens (methyl- and propyl-) at 1000 mg kg–1 per day. Data were analysed using a nonparametric one-way ANOVA followed by Dunnett's test for multiple comparisons. The ovaries were excised at postnatal Day 8 and fixed and stained with haematoxylin and eosin for histological analysis. The number of follicles were counted and classified into primordial, primary, or secondary stages via the largest serial sections at the centre of the ovary. Results showed that the total number of follicles and number of primordial follicles were increased by methyl- and propyl-parabens compared with the vehicle. Taken together, these results indicated that methyl- and propyl-parabens may have an estrogenic effect that disrupts the ovarian follicle development necessary for reproductive functions in a neonatal female rat model.
136 USE OF NANOFLUIDIC RT QUANTITATIVE POLYMERASE CHAIN REACTION TO STUDY VARIATION IN GENE EXPRESSION BETWEEN SINGLE BOVINE OOCYTE AND BLASTOCYST SAMPLES
Authors: Morrill B. H. , Sessions B. R. , White K. L. , Isom S. C.
Published: December 2011

Most research into gene expression of oocyte and pre-implantation-stage embryos has been on pooled samples. Little work has been done at the single oocyte or early embryo levels. Our hypothesis was that a large amount of variation exists in relative transcript abundance between individual oocytes/embryos, which may ultimately be associated with inherent developmental potential. Because most studies to date have been on pooled samples, only very limited data have been presented that explain whether such variation exists, or if most oocytes/embryos within a pool are similar in gene expression. To generate preliminary information about the amount of variation in gene expression that exists between single oocytes and blastocyst-stage embryos, 4 single oocyte and 8 single blastocyst samples were compared with pooled oocyte samples of 10 and 20 and a pooled blastocyst sample of 10. Oocyte maturation and embryo production were performed according to established protocols (see Aston et al. 2010 Cell Reprog.). The 24 genes selected for use in this analysis consisted mostly of pluripotency genes, imprinted genes and housekeeping genes. To analyse such small samples we used nanofluidic BioMark® quantitative PCR (qPCR) technology from Fluidigm (San Francisco, CA, USA). Complementary DNA was generated and preamplified by adding the samples listed above directly to a reverse-transcription/preamplification mix according to a prevalidated protocol from Fluidigm. Samples were run in duplicate on a single 48.48 microfluidic chip using intercalating dye qPCR technology. Each primer set was run in duplicate as well, resulting in 4 data points for each sample and primer set combination. Data were analysed using the relative standard curve method. We were able to produce preliminary data on how reproducible data generation is with this platform on single oocyte and blastocyst samples. Correlations between assay and sample replicates were high (R2 = 0.998 and R2 = 0.967, respectively). Among the oocyte samples, a fold change difference of 2 or more was found in 9 of the 24 primer sets. A similar analysis of the blastocyst samples yielded fold changes over 2 for 12 of the 24 primer sets. These preliminary data suggest that the use of nanofluidic qPCR technology may be useful for the study of gene expression variation among single oocyte and blastocyst samples.
Funding was provided to S. C. Isom through the Utah Agricultural Experiment Station (program project #UTA00151).
137 DIFFERENTIAL GENE EXPRESSION OF IN VITRO-MATURED BOVINE OOCYTES WITH OR WITHOUT A POLAR BODY
Authors: Pereira M. M. , Wohlres-Viana S. , Sales J. N. S. , Camargo A. R. , Quintão C. C. R. , Rabelo N. C. , Carvalho B. C. , Viana J. H. M. , Camargo L. S. A. , Guimarães M. F. M. , Iguma L. T.
Published: December 2011

Oocyte competence is associated with the amount of transcripts stored in the ooplasm and oocyte ability to extrude polar bodies (PB). To our knowledge, however, no data comparing mRNA levels between bovine oocytes maturated in vitro with or without PB are available. The aim of the present study was to compare the relative abundance of transcripts of glucose transporter 1 (GLUT1), insulin-like growth factor 1 receptor (IGF1R), insulin-like growth factor 2 receptor (IGF2R), growth differentiation factor-9 (GDF9) and aquaporin 3 (AQP3) genes between oocytes with and without PB (PB and NPB groups, respectively) following in vitro maturation. Immature bovine oocytes were obtained by follicular aspiration and matured in TCM-199 (Gibco Life Technologies, New York, NY, USA) containing 10% of oestrus cow serum and 20 μg mL–1 of FSH (Pluset, Serono, Italy) for 24 h under 5% CO2 in air at 38.5°C. Subsequently, oocytes were visually classified according to the presence or absence of PB and then denuded and rapidly frozen in liquid nitrogen. Three pools of 10 oocytes for each group were subjected to total RNA extraction using the RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions and treated with DNase. Reverse transcription and cDNA amplification were performed using the TransPlex Complete Whole Transcriptome Amplification Kit (WTA2, Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. Relative abundance of the target transcripts was performed by quantitative RT-PCR (Applied Biosystems Prism 7300 Sequence Detection Systems, Foster City, CA, USA) using a mixture of SYBR® Green PCR Master Mix (Applied Biosystems), 200 ng of cDNA, nuclease-free water and specific primers for each reaction. Expression of the β-actin gene was used as an endogenous reference. Relative gene expression analysis was performed using the software REST© 2005 using the Pair Wise Fixed Reallocation Randomization Test©. The relative expression values are presented as mean ± standard error. The relative abundance of GLUT1 (0.81 ± 0.07), IGF2R (0.72 ± 0.07) and GDF9 (0.82 ± 0.10) genes was lower (P < 0.05) for NPB oocytes. There was no difference (P > 0.05) in relative abundance between PB and NPB groups for the other genes. The results suggest that the amount of some transcripts stored in the matured ooplasm is associated with the presence of PB.
The authors acknowledge FAPEMIG, CNPq and Innovation Network Project on Animal Reproduction (01.07.01.002).
144 EXPRESSION OF ACETYL COENZYME A CARBOXYLASE α (ACCα) IN FELINE, CANINE AND PORCINE OOCYTES
Authors: Bolden C. , Abdela W. , Samuel T. , Simon L. , Wirtu G.
Published: December 2011

High lipid content in in vitro–derived embryos of several species is associated with poor developmental potential and cryosurvival of oocytes. Lipid content of oocytes varies among species and embryonic lipid content is further influenced by the culture environment. In spite of the variation among species, the relative contribution of de novo lipid synthesis during oocyte maturation or subsequent embryonic development is unknown. In the present study, we evaluated the expression of acetyl coenzyme A carboxylase (ACCα), the key rate-limiting enzyme of lipogenesis, in oocytes of 3 species with high lipid content. Ovaries of dogs and cats were collected from a local veterinary clinic and those of pigs from an abattoir. Cat and dog oocytes were recovered by the slicing method of the ovaries. Porcine oocytes were recovered by aspiration of ovarian follicles. Immediately after collection, oocytes were fixed for 30 min using a solution of 10% neutral buffered formalin. The ACCα enzyme expression was evaluated in porcine, cat and dog oocytes by immunofluorescence using a goat anti-human ACCα primary antibody followed by fluorescein isothiocyanate-conjugated anti-goat secondary antibody. The study was replicated, with negative controls, 3 times using ≥30 oocytes per species. Messenger RNA expression of ACCα gene was also evaluated in pig oocytes. The RNA was isolated from fat tissue, pooled good-quality oocytes (n = 30), pooled poor-quality oocytes (n = 30) and cumulus cells using the RNeasy Micro kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Complementary DNA was synthesised from 200 ng of RNA using Quantitect reverse-transcription kit (Qiagen) according to the manufacturer's instructions. Real-time PCR assays were carried out in duplicate and expression of ACCα mRNA levels relative to fat was determined. Oocytes and cumulus investment in cats, dogs and pigs strongly expressed ACCα. Expression of the protein was uniformly distributed through the entire ooplasm. The mRNA expression of ACCα in good- and poor-quality oocytes and cumulus cells relative to fat tissue was 11.5, 1.4 and 40.1%, respectively. Further studies are warranted on the dynamics of expression of ACC during in vitro maturation and the functional activity of the enzyme.
We extend our appreciation to the Alabama Animal Alliance Inc. (Montgomery) for providing us with dog and cat ovaries; the Lambert-Powell Meats Laboratory of Auburn University for donating pig ovaries; and Dr. James Webster, Dr. Tsegaye Habtemariam and Dr. Abdalla Eljack for administrative support.
162 EFFECT OF THE SPERM SELECTION WITH ISOLATE® OR PERCOLL® ON SPERM QUALITY AND IN VITRO BOVINE EMBRYO DEVELOPMENT FOR FROZEN - THAWED SEXED AND NONSEXED SEMEN
Authors: Bó G. A. , Rodriguez Villamil P. , Moreira G. , Garcia Gomez M. E. , Fernandez Taranco M. , Wei H.
Published: December 2011

The aim of this study was to compare the effects of 2 different commercially available spermatozoa separation techniques, Isolate® (Irving-Scientific, Santa Ana, CA, USA) and Percoll® (Nutricell, São Paulo, Brazil), on sperm quality and in vitro embryo production using sexed and nonsexed semen. Oocytes (n = 5046) were obtained from slaughterhouse ovaries and fertilized with frozen-thawed sexed or nonsexed semen from the same 4 Holstein bulls. The experiment was done in 10 replicates, with all treatment groups included. Sperm quality (motility, concentration, morphology and membrane integrity) was evaluated and compared before and after sperm selection by the 2 methods. Oocytes were maturated in TCM-199 supplemented with 0.4% of BSA for 24 h in a controlled atmosphere and then selected and randomly allocated into 4 different groups. Group 1: oocytes fertilized with sexed semen selected by Percoll®; Group 2: oocytes fertilized with sexed semen selected by Isolate®; Group 3: oocytes fertilized with nonsexed semen selected by Percoll®; Group 4: oocytes fertilized with nonsexed semen selected by Isolate®. Fertilization was performed in Fert-TALP medium for 18 h under the same conditions as maturation. Presumptive zygotes were cultured for 7 days in SOF medium in a 39°C humidified incubator with 5% CO2, 5% O2 and 90% N2. Cleavage and blastocyst rates were evaluated on Day 2 and 7, respectively, after fertilization. Proportional data were transformed by square root and then analysed by ANOVA, with type of semen and sperm selection method as the main effects. Regardless of the sperm selection technique, sperm motility and percentage of normal sperm increased (P < 0.005) from the initial post-thaw parameters. For nonsexed semen, Percoll® gradient increased the recovery rate (i.e. final concentration/initial concentration × 100; 57.3 ± 2.7) compared with Isolate® (46.0 ± 1.8; P < 0.05). Furthermore, sperm selected by Isolate® presented significant improvements compared with Percoll® gradient on membrane integrity of sexed (41.0 ± 0.6 vs 38.8 ± 0.8) and nonsexed semen (60.8 ± 1.6 vs 58.8 ± 0.5; P < 0.05). Finally, blastocyst production rates were higher (P < 0.05) for sexed (Group 2: 14.0 ± 1.0) or nonsexed semen (Group 4: 22.0 ± 1.1) selected by Isolate® than for sexed (Group 1: 10.5 ± 1.5) or nonsexed semen (Group 3: 17.0 ± 2.1) selected with Percoll®. In conclusion, selection of both sexed and nonsexed semen for IVF with Isolate® resulted in higher quality sperm and higher embryo production rates than Percoll®.
164 INFLUENCE OF LINEAGE OF OOCYTE DONOR ON THE YIELD AND MORPHOLOGY OF OOCYTES RECOVERED BY ULTRASOUND-GUIDED FOLLICULAR ASPIRATION IN NELLORE COWS
Authors: Martins A. , Calegari R. S. , Paschoal D. M. , Souza D. G. , Sudano M. J.
Published: December 2011

Several factors exert considerable influence on oocyte production and quality, such as donor age, nutrition, season and others. However, very few studies have been done to verify the effects of breed on the quantity or quality of oocytes, or both. In Brazil, the Nellore breed has been developed as a result of many years of selective breeding from 6 major ancestor bulls imported from India in the 1960s. These animals have characterised their respective lineages, as evidenced by the well-defined genetic traits. This study was carried out to investigate the influence of the genetic lineage of oocyte donors on the number and quality of oocytes obtained through ultrasound-guided follicular aspiration in Nellore cows derived from 2 lineages of bulls [Karvadi (K) and Taj Mahal (T)]. Both maternal (Km and Tm) and paternal (Kp and Tp) lineages were investigated. Oocyte aspirations were repeatedly performed with an aspiration interval of 15 days without the superovulation procedure in 56 donor females over a period of 2 years. At the beginning of the experiment, the animals were on average 78 ± 12 months old with a body condition score of 3.6 ± 0.08 (on a 5-point scale). Recovered cumulus–oocyte complexes were counted, morphologically examined and classified into 7 categories (grades I to VII) according to the number of layers of the cumulus–oocyte and the appearance of the cytoplasm. Data were analysed using ANOVA and Tukey's test after transformation into log(x + 1). Values are reported as means ± s.e.m. and differences (P > 0.05) were taken as significant. The mean number of oocytes retrieved from donors of lineage Tp-Tm was higher (n = 9; 28.23 ± 1.92; P < 0.05) than that obtained for lineages Kp-Tm (n = 11; 21.34 ± 1.32), Kp-Km (n = 15; 21.28 ± 1.73) and Tp-Km (n = 21; 16.72 ± 1.31). There was no significant difference in the mean number of recovered oocytes between donors of lineages Kp-Tm and Kp-Km; however, lineage Tp-Km yielded a lower number of oocytes. Donors of lineage Kp yielded more (P < 0.05) oocytes of grade III than lineage Tp (10.11 ± 0.66 vs 8.79 ± 0.58, respectively). These lineages produced a higher mean number of oocytes graded as I, II and III (3.14 ± 0.21, 4.93 ± 0.33 and 10.11 ± 0.66 vs 3.19 ± 0.21, 5.59 ± 0.44 and 8.79 ± 0.58, respectively) than any other categories. However, more oocytes (P < 0.05) of grades I, II and III were obtained for lineage Tm (3.64 ± 0.25, 5.9 ± 0.42 and 11.67 ± 0.67) than for lineage Km (2.85 ± 0.19, 4.92 ± 0.40 and 7.73 ± 0.56, respectively), with similar results for oocytes of grades IV, V, VI and VII. In conclusion, the genetic lineage of the oocyte donor influenced the production and quality of oocytes obtained through ultrasound-guided follicular aspiration in Nellore cows. Selection of donor cows based on their oocyte morphology and availability can further improve IFV programs.
168 CORRELATION OF DEVELOPMENT KINETICS AND SEX OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Buzzo A. R. , Pupulim A. R. , Mazucheli J. , Meirelles F. V. , Emanuelli I. P.
Published: December 2011

Approaches to improve the culture medium for in vitro production (IVP) of bovine embryos have been continuous because of the high commercial demand and a portion of this attempts the production of female cattle (dairy cows and stud cattle). However, in some embryonic in vitro culture systems, the development kinetics is faster in male than in female embryos (Avery 1992 Mol. Reprod. Dev. 32, 265–70; Xu 1992 Mol. Reprod. Dev. 31, 249–50). The aim of this work was to relate the kinetics of blastocyst expansion with the production rates of male and female embryos. Cumulus–oocyte complexes (n = 917; classes I and II) of cows from a slaughterhouse were matured with TCM-199 bicarbonate and 10% FCS (38.5°C, 5% CO2) for 24 h and fertilized with frozen-thawed semen in TALP-IVF medium for 18 h. Presumptive zygotes were culture in SOF medium supplemented with 10% FSB (5% O2, 38.5°C). Seven days after IVF, embryos were divided in 2 groups according to their kinetic stage of development: nonexpanded blastocysts (n = 175), or hatched and expanded blastocysts (n = 146). Hence, embryos were individually frozen in LN and stored in cryotubes. After thawing, Proteinase K (16 mg mL–1) was added to each tube and the tubes were incubated for 60 min at 37°C. Proteinase was denatured at 98°C for 10 min and the contents of each tube were divided into 2 samples (A and B) and subjected to the PCR technique. Two pairs of primers for the specific sequence of the Y chromosome were used to amplify the sequence of 210 and 250 bp for the male bovine and 1 pair of primers was used for the autosomal bovine sequence with a 280-bp fragment. Female embryos with a 280-bp product were observed in sample A and none were observed in sample B. The presence of 2 amplicons (280 and 210 bp) in sample A and 1 amplicon of 250 bp in sample B indicated that the embryo was male. A chi-square test was used to evaluate homogeneity. An analysis of the percentage of males and females between the experimental groups was performed by logistic regression and significance was considered when P < 0.05. There was no difference in the proportions of males and females in the nonexpanded blastocyst group (49.71 and 50.29%; P > 0.05). In the hatched and expanded blastocyst group, the proportion of males (65.75%) was statistically different from the proportion of females (34.25%); that is, the chance of the embryo being male was twice as high (P < 0.0038). These results suggest that there is a difference in the kinetics of embryo development between male and female embryos and that blastocyst expansion can point that out. In vitro culture media with FCS support the development of expanded male blastocysts. Further research in culture medium modifications (FCS, the energy source, amino acids and others) are needed to respond to the trend in the production of sex-defined embryos.