In Vitro Embryo Production Research Articles

148 EXPRESSION OF ARTEMIN IN THE BOVINE ENDOMETRIUM, UTERINE FLUID, AND EARLY EMBRYOS

Artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family, is expressed in human and mice pre-implantation embryos and reproductive tract (Li et al. 2009 FEBS Lett. 583; Kawamura et al. 2012 PLoS One 7). In mice, artemin promotes in vitro embryo development and decreases apoptosis (Li et al. 2009 FEBS Lett. 583). The presence of artemin in cattle embryos and endometrium, however, is unknown. In this work we analysed artemin expression in bovine blastocysts and endometrium by immunohistochemistry and in uterine fluid (UF) by Western blot (WB). Briefly, Day-6 in vitro-produced (IVP) embryos (n = 50) were nonsurgically transferred to the uterus of heifers (n = 10, 50 IVP embryos per heifer) at nonconsecutive oestrus cycles. On Day 8, embryos and their corresponding diluted UF were flushed; blastocysts that developed entirely in vitro were also collected. In addition, endometrial samples were collected on Day 8 from slaughtered females that were embryo transferred (n = 6) and sham transferred (n = 6) on Day 5. Artemin localization was investigated in blastocysts and in endometrial samples, using immunohistochemical staining methods described elsewhere (Muñoz et al. 2012 J. Proteome Res. 11; Gómez et al. 2014 Reproduction pii: REP-14–0304). The signal-strength comparisons between uterus-exposed and IVP blastocysts were analysed using the software Confocal Uniovi Image-J. Quantification of WB protein bands was achieved by computer-assisted densitometry using Image-J software. Artemin was detected, with similar intensity, in the inner cell mass and trophectoderm from both uterus-developed and IVP blastocyst. All embryos analysed expressed artemin. The signal intensity and staining pattern observed did not differ between uterus-exposed and IVP blastocysts. In the endometrium, the most intense staining for artemin was localised to the apical sites in the luminal epithelium and in the glandular epithelium of superficial glands. There was also diffuse staining in the stroma and deep uterine glands. The uterine region and pregnant or cyclic status did not affect the artemin staining pattern. Artemin was detected by WB in all UF samples analysed (embryo transferred N = 10, sham transferred N = 10). However reliable quantitation of artemin by WB was unfeasible due to the broad dynamic range of artemin expression through samples. In conclusion, our results demonstrate the presence of artemin in bovine uterine endometrium and UF, and embryos during early development. As shown in mice, it is feasible that artemin might exert an autocrine/paracrine role during early embryo development in the cow. The study received grant support: Spanish Ministry of Science and Innovation (MINECO, project AGL2012–37772 and FEDER). M. M. was supported by grant MICINN-RYC08–03454. The authors are members of the COST Action FA1201 Epiconcept.

43 NEW METHOD FOR ONE-STEP WARMING/IN-STRAW CRYOPROTECTANT DILUTION FOR IN VITRO-PRODUCED BOVINE BLASTOCYSTS AFTER VITRIFICATION WITH THE CRYOLOGIC VITRIFICATION METHOD

Vitrification is considered an alternative to slow-rate freezing to cryopreserve in vitro-produced (IVP) bovine embryos. However, the use of vitrified IVP embryos for embryo transfer under field conditions is difficult because of the requirements of the current thawing protocols. The objective of this study was to develop a simple one-step warming/in-straw cryoprotectant dilution procedure for IVP bovine blastocysts that were vitrified using the cryologic vitrification method. In this study, 109 Day-7 IVP blastocysts were subjected to vitrification using the conventional fibreplugs (groups of 5 embryos were loaded in 3 mL of vitrification medium). Warming was performed in one-step in MS1 (0.25 M sucrose in BV = TCM 199-Hepes + 20% FCS) either using a 4-well plate for 5 min (control group) or in a new system that allowed in-straw cryoprotectant dilution designed to avoid losses of embryos and to maintain the temperature required during this procedure. This new system is composed of an adaptor with a wider opening that is coupled to the French straw and a heated metal chamber to protect and keep the straw at 41°C. Warmed embryos were washed and subsequently cultured in mSOFaaci + 6 gL–1 BSA + 10% FCS for 48 h. Re-expansion (at 2, 24, and 48 h) and hatching rates (at 24 and 48 h) were recorded. Data were analysed by ANOVA and are presented as LSM ± standard error. Embryo survival rates of embryos warmed by the one-step warming/in-straw cryoprotectant dilution procedure did not differ from the control group (see Table 1). These results suggest that the cryologic vitrification method combined with our warming system for in-straw cryoprotectant dilution may be used for direct embryo transfer under field conditions. Table 1.Embryo survival rates of in vitro-produced embryos vitrified by the cryologic vitrification method and warmed by the new one-step warming/in-straw cryoprotectant dilution procedure This study received grant support: INIA-RTA 2011–0090 and FEDER. M. Muñoz was supported by grant MICINN-RYC08-03454, and B. Trigal by a grant from Cajastur. The authors are members of the COST Action FA1201 Epiconcept.

Differences in amniotic amino acid concentrations between pregnancies obtained with transfer of vitrified thawed in vitro–produced embryos and with natural mating in sheep

In vitro embryo production (IVP) and cryopreservation are associated with a high incidence of pregnancy complications and fetal abnormalities that may be linked with alterations of placental development. The amniotic fluid is partly derived from the transport of water and solutes across the placenta and provides the fetus with amino acids (AAs), which are the building blocks for biomolecules involved in physiological growth and development. To better understand the anomalies associated with IVP pregnancies, the present study was conducted to test the hypothesis that amniotic concentrations of AAs differ in pregnancies derived from vitrified/thawed (V/T) IVP embryos compared with gestations obtained with natural mating (NM) in sheep. Amniotic fluid was sampled in ewes that were pregnant after transfer of V/T IVP embryos and that had conceived with NM between Days 60 and 65 (V/T, n = 6; NM, n = 11) and between Days 80 and 85 (V/T, n = 5; NM, n = 14) of gestation via ultrasound-guided amniocentesis. Concentrations of 16 AAs in the amniotic fluid were measured using high-performance liquid chromatography. From Days 60 to 65 of gestation, concentrations of cystine, phenylalanine, and isoleucine were lower in V/T compared with NM ewes. From Days 80 to 85 of pregnancy, the mean concentrations of cystine and lysine were lower in the V/T versus NM groups. The total AA concentration per ewe was similar between the groups from Days 60 to 65 and 80 to 85 of gestation and decreased by 55% from Days 60 to 65 and 80 to 85 of gestation in all ewes. The most abundant AA from Days 60 to 65 of gestation was alanine in both groups, whereas from Days 80 to 85, the most abundant AAs were alanine in NM and glycine in V/T ewes; cystine was the less abundant detectable AA in all ewes at both stages of gestation. Results report that V/T IVP embryos have decreased concentrations of individual AAs in the amniotic fluid during the second trimester of gestation possibly because of an impaired placental vasculogenesis or because of a reduced placental transport. These novel findings are relevant to unravel the mechanisms responsible for the issues of pregnancies achieved with the transfer of IVP and cryopreserved embryos.

The effects of superoxide dismutase addition to the transport medium on cumulus–oocyte complex apoptosis and IVF outcome in cats (Felis catus)

The aim of the present study was to examine the effects of superoxide dismutase (SOD) addition to the ovary transport medium (4°C, 3–72h) on ovarian cell viability and apoptosis and in vitro embryo production (IVEP) in domestic cats. The ovaries collected from 76 mixed-breed domestic queens were randomly assigned to the control or SOD-treated groups and incubated for 3, 24, 48 or 72h. The ovaries were then subjected to the following: (1) fixed in formalin to assess the incidence of apoptosis (fragmented DNA in situ detection kit), (2) stored at −196°C in liquid nitrogen to evaluate the expression of the pro-apoptotic Bax gene and the anti-apoptotic Bcl-2 gene (RT-PCR), and (3) used to obtain the cumulus–oocyte complexes (COCs) in order to test the cell viability (carboxyfluorescein or trypan blue staining) and IVEP. The incidence of apoptosis appeared to be higher in the control compared with the SOD-treated ovaries. The ovarian expression of Bax was lower and the Bcl-2 expression was higher in the SOD-treated group compared with the control group. The presence of SOD in the transport medium increased the viability of COCs and IVEP compared with the control medium. In summary, the supplementation of the ovary transport medium with SOD reduced cellular apoptosis and enhanced COC survival and IVEP in domestic cats.

The interval between the emergence of pharmacologically synchronized ovarian follicular waves and ovum pickup does not significantly affect in vitro embryo production in Bos indicus, Bos taurus, and Bubalus bubalis

The aim of the present study was to determine the optimal phase of the follicular wave to perform ovum pickup (OPU) for in vitro embryo production (IVEP) in various genetic groups. For this purpose, 27 heifers—nine Bos taurus (Holstein), nine Bos indicus (Nelore), and nine Bubalus bubalis (Mediterranean)—were maintained under the same nutritional, management, and environmental conditions. Heifers within each genetic group were submitted to six consecutive OPU trials with 14-day intersession intervals, at three different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5 after follicular wave emergence), in a 3 × 3 crossover design. When OPU was performed at different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5), no differences were found in the percent of oocytes recovered (70.5 ± 3.1%, 75.0 ± 3.1%, 76.0 ± 3.2%, respectively; P = 0.41) or blastocyst production rates (19.4 ± 2.9%, 16.6 ± 2.9%, 15.9 ± 2.6%, respectively; P = 0.36). Comparing genetic groups, B indicus showed a higher blastocyst rate (28.3a ± 2.8%; P < 0.01) than B taurus and B bubalis (14.1b ± 2.9% and 10.2b ± 2.0%, respectively). However, only B indicus heifers showed a variation in the number of visualized follicles and the total and viable oocytes along consecutive OPU sessions. In conclusion, different phases of the pharmacologically synchronized ovarian follicular wave did not affect OPU-IVEP in B indicus, B taurus, and B bubalis heifers. Additionally, B indicus heifers showed greater OPU-IVEP efficiency than did the other genetic groups, under the same management conditions.

Current status of in vitro embryo production in sheep and goats.

Sheep and goat production is an important economic activity in Spain with an increasing interest in milk production. Multiovulation and Embryo Transfer (MOET) and In vitro Embryo Production (IVEP) are assisted reproductive technologies aimed at increasing the genetic diffusion of females. In vitro embryo production is a multi-step methodology comprising the following procedures: (i) In vitro Maturation (IVM) of oocytes recovered directly from the follicles, (ii) In vitro Fertilization (IVF) or co-incubation of capacitated spermatozoa with in vitro matured oocytes and (iii) In vitro culture (IVC) of zygotes up to the blastocyst stage. In vitro embryo production from oocytes recovered from prepubertal females is called JIVET (Juvenile in vitro Embryo Transfer) and allows shortened generation intervals and increased genetic gain. Embryo production together with embryo cryoconservation would allow large-scale embryo marketing, a pathogen-free genetic movement and easier and cheaper germplasm commercial transactions. Commercial Embryo activity in small ruminants is low compared to cows in the European Union (data from the European Embryo Transfer Association) and in the world (data from the International Embryo Transfer Association). There is less IVEP research in small ruminants compared to other livestock species. The aim of this review was to provide an overview of the current status of IVEP of small ruminant with an emphasis on (i) description of the main methodologies currently used for IVM, IVF and IVC of embryos (ii) comparing procedures and outputs from JIVET and IVEP of adult females and (iii) the future research perspectives of this technology.

In vitro ovine embryo production: the study of seasonal and oocyte recovery method effects.

Background:To current knowledge, different oocyte's recovery method and various seasons have profound impact on in vitro embryo production (IVEP).Objectives:The aim of this study was to define an efficient recovery method for oocytes harvesting from slaughterhouse material in different seasons, and their effects on IVEP yield.Materials and Methods:Ovaries from slaughtered ewes in breeding season (BS) and non-breeding season (NBS) were collected from a local abattoir. The oocytes were recovered through aspiration, centrifugation (ORC), puncture and slicing, and categorized into three classes (I, oocytes with more than three layers of cumulus cells; II, less than three layers with damaged cumulus cells; III, denuded oocytes). After cultivation in TCM 199 for 24 hours, matured oocytes were subjected to in vitro fertilization (IVF) and in vitro culture (IVC). The oocyte recovery using ORC in BS and NBS was significantly higher (P < 0.05) compared with other recovery methods.Results:No significant dissimilarities in the proportion of oocytes reaching M-II stage were recorded when using different oocyte recovery methods in different seasons. Aspiration resulted in lower (P < 0.05) proportion of class I (BS, 60.0 ± 2.1; NBS, 51.1 ± 2.1) compared to ORC (BS, 82.0 ± 1.2; NBS, 70.0 ± 1.2), slicing (BS, 80.0 ± 2.1; NBS, 71.0 ± 1.4) and puncture (BS, 80.0 ± 1.5; NBS, 72.0 ± 2.0). Monospermy and blastocyst development rates were significantly higher using ORC than other recovery techniques in both BS and NBS. More oocytes with high quality, greater blastocyst development and oocyte recovery rates were achieved in BS.Conclusions:The results revealed that oocytes harvesting technique and season are effective in the rate of cleavage and blastocysts’ development, and suggest that despite same meiotic resumption rate in all treatments, it would be better to use ORC.

Plasma anti-mullerian hormone: an endocrine marker for in vitro embryo production from Bos taurus and Bos indicus donors

The aim of this study was to evaluate the association between plasma anti-mullerian hormone (AMH) concentration and in vitro embryo production (IVP) from Bos taurus (Holstein) and Bos indicus (Nelore) donors. A total of 59 Holstein (15 prepubertal heifers aged 8–10 mo, 15 cyclic heifers aged 12–14 mo, 14 lactating cows, and 15 nonlactating cows) and 34 Nelore (12 prepubertal heifers aged 10–11 mo, 10 prepubertal heifers aged 21–23 mo, and 12 cyclic heifers aged 24–26 mo) females were enrolled. All females underwent an ovum pick-up (OPU), without previous synchronization of the follicular wave, and IVP procedure. Immediately before the OPU procedure, blood samples were collected for subsequent AMH determination. A positive correlation was observed between the plasma AMH and number of in vitro embryos produced from Holstein (r = 0.36, P < 0.001) and Nelore (r = 0.50, P = 0.003) donors. For additional analyses, donors within each genotype were classified into 1 of 2 AMH categories (low or high) according to the average AMH concentration for each genotype. The results revealed that females classified as having high AMH presented a greater number of visible aspirated follicles (Holstein: 20.9 ± 1.5 vs 13.6 ± 0.9, P < 0.0001; Nelore: 54.3 ± 6.1 vs 18.6 ± 2.1, P < 0.0001) and a greater number of recovered cumulus-oocyte complexes (Holstein: 17.3 ± 1.5 vs 9.0 ± 0.9, P < 0.0001; Nelore: 45.3 ± 6.4 vs 13.4 ± 1.7, P < 0.0001). However, there was no difference in the blastocyst production rate (Holstein: 20.6% ± 4.0% vs 19.8% ± 4.2%, P = 0.60; Nelore: 33.7% ± 6.5% vs 27.4% ± 5.5%, P = 0.41, high and low AMH, respectively). Moreover, donors classified as having high AMH yielded a greater number of embryos produced per OPU (Holstein: 3.0 ± 0.7; Nelore: 7.0 ± 1.7) compared with those classified as having low AMH (Holstein: 1.2 ± 0.3, P = 0.04; Nelore: 2.2 ± 0.5, P = 0.007). In conclusion, although the plasma AMH concentration did not alter the ability of the cumulus-oocyte complex to reach the blastocyst stage, the AMH concentration in plasma can be an accurate endocrine marker for the in vitro embryo yield from either B. taurus (Holstein) or B. indicus (Nelore) donors. Therefore, AMH is a promising tool to enhance the overall efficiency of OPU–IVP programs in the field as a selective criterion for high embryo producing donors.

Prediction of pregnancy viability in bovine in vitro-produced embryos and recipient plasma with Fourier transform infrared spectroscopy

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared (FTIR) metabolomics to predict pregnancy outcome. Individually cultured, in vitro-produced (IVP) blastocysts were transferred to recipients as fresh and vitrified-warmed. Spent CM and plasma samples were evaluated using FTIR. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the receiver operator characteristic curve (AUC). Within all IVP fresh embryos (birth rate=52%), high AUC were obtained at birth, especially with expanded blastocysts (CM: 0.80±0.053; plasma: 0.89±0.034). The AUC of vitrified IVP embryos (birth rate = 31%) were 0.607±0.038 (CM, expanded blastocysts) and 0.672±0.023 (plasma, all stages). Recipient plasma generally predicted pregnancy outcome better than did embryo CM. Embryos and recipients with improved pregnancy viability were identified, which could increase the economic benefit to the breeding industry.

Impaired placental vasculogenesis compromises the growth of sheep embryos developed in vitro.

To evaluate how assisted reproductive technologies (ART) affect vasculogenesis of the developing conceptus, we analyzed placental and fetal development of in vitro-produced (IVP) sheep embryos. Pregnancies produced by ART carry increased risk of low birth weight, though what causes this risk remains largely unknown. We recently reported that developmental arrest of sheep conceptuses obtained by ART is most pronounced when the cardiovascular system develops (Days 20-30 of development). A total of 86 IVP blastocysts (2-4 per ewe) were surgically transferred to 30 recipient sheep 6 days after estrus; 20 sheep were naturally mated (control). Conceptuses were recovered from sheep at Days 20, 22, 26, and 30 of gestation and morphologically evaluated. Then, the conceptuses and part of their placentae (chorion-allantois) were fixed for histological and immunohistochemical analysis and snap-frozen in liquid nitrogen for subsequent mRNA expression analysis. Results demonstrate that the cardiovascular systems of sheep IVP conceptuses were severely underdeveloped. Pericardial and placental hemorrhages were noted in a majority (5/7) of the dead embryos. In the surviving IVP embryos, the expression of angiogenetic factors was reduced at Day 20. The placental vessels were underdeveloped on Days 20 and 22 (P < 0.05), though placental vasculogenesis was successfully completed on subsequent days. However, low vessel number persisted at Days 26 and 30 (4.6 vs. 5.9 and 6.64 vs. 8.70 per field, respectively; P < 0.05) together with reduced vessel diameter at Day 26 (46.89 vs. 89.92 μm; P < 0.05). In vitro production of sheep embryos induced severely impaired vasculogenesis early in gestation. This may lead to developmental programing problems, such as intrauterine growth restriction of the fetus, resulting in long-term health consequences for the offspring, such as cardiovascular diseases.

Repeated hormonal treatment and laparoscopic ovum pick-up followed by in vitro embryo production in goats raised in the tropics

The objectives of this study were to evaluate the ovarian response of oocyte donor goats which underwent seven repeated ovulation hormonal treatments and laparoscopic ovum pick-up (Experiment 1), and to compare the In vitro production (IVP) of embryos from goats hormonally treated for eighth time to IVP of embryos from females with a single hormonal treatment (Experiment 2). In Experiment 1, 12 goats were subjected to 7 repeated hormonal treatments and laparoscopic ovum pick-up (LOPU) every 2–3 weeks, and the following variables were recorded: number and size of punctured follicles, number of recovered cumulus-oocyte complexes (COCs), recovery rate and COCs quality. In Experiment 2, IVP of embryos from goats hormonally treated for eighth time (repeated-treated group, RT) was compared with IVP of embryos from 10 other goats with only one hormonal treatment (single-treated group, ST). In this step, in addition to variables observed in Experiment 1, cleavage, blastocyst rates at Days 7 and 8 of culture, hatched rate and the number of blastomeres per embryo were also evaluated. In Experiment 1, when comparing LOPU sessions, no difference (P>0.05) was verified for number of punctured follicles, number of recovered COCs and recovery rate. However the percentage of large follicles was different (P<0.05) between LOPU 1 (27.7±10.5) and 7 (12.4±12.1), the latter being similar to sessions 2 (13.9±10.9), 3 (13.4±7.9), 4 (13.2±10.7), 5 (22.4±14.7) and 6 (17.7±15.8). The percentage of COCs suitable for IVM was lower (P<0.05) for LOPU 1 (80.6%), 2 (82.4%) and 3 (79.7%) than LOPU 6 (91.3%) and 7 (95.2%). In Experiment 2, total number of follicles aspirated/goat and total number of COCs recovered were similar (P>0.05) for RT (18.0±5.8 and 12.3±3.7, respectively) and ST (19.4±7.8 and 15.8±7.7, respectively) groups. However, the recovery rate was lower (P<0.05) in the RT vs the ST group (68.5% and 81.4%). The percentage of small, medium and large follicles was similar (P>0.05), respectively, for RT (28%, 55% and 17%) and ST (20%, 65% and 15%) groups. The percentage of COCs suitable for IVM had no difference (P>0.05) for RT (96.6%) and ST (93.7%) groups. No significant difference was observed between RT and ST groups for: cleavage rate (68.6% vs 67.0%), blastocyst rate at Days 7 (27.9% vs 31.8%) and 8 (27.9% vs 34.1%), hatched rate (33.3% vs 53.3%), and blastomeres/embryo (252.9 vs 229.8). In conclusion, in goats raised in tropical climate, the IVP of embryos from oocytes obtained by LOPU could be an efficient and suitable method for rapid propagation of genetically superior animals. Donor goats, even after repeated hormonal treatments followed by LOPU, maintained the ovarian response for oocyte and embryo production, becoming close to reality the term “oocyte permanent donors”.

Developmental Competence of Bovine Early Embryos Depends on the Coupled Response Between Oxidative and Endoplasmic Reticulum Stress1

The stress produced by the coupling of reactive oxygen species (ROS) and endoplasmic reticulum (ER) has been explored extensively, but little is known regarding their roles in the early development of mammalian embryos. Here, we demonstrated that the early development of in vitro-produced (IVP) bovine embryos was governed by the cooperative action between ROS and ER stress. Compared with the tension produced by 5% O2, 20% O2 significantly decreased the blastocyst formation rate and cell survival, which was accompanied by increases in ROS and in levels of sXBP-1 transcript, which is an ER stress indicator. In addition, treatment with glutathione (GSH), a ROS scavenger, decreased ROS levels, which resulted in increased blastocyst formation and cell survival rates. Importantly, levels of sXBP-1 and ER stress-associated transcripts were reduced by GSH treatment in developing bovine embryos. Consistent with this observation, tauroursodeoxycholate (TUDCA), an ER stress inhibitor, improved blastocyst developmental rate, trophectoderm proportion, and cell survival. Moreover, ROS and sXBP-1 transcript levels were markedly decreased by supplementation with TUDCA, suggesting a possible mechanism governing the mutual regulation between ROS and ER stress. Interestingly, knockdown of XBP-1 transcripts resulted in both elevation of ROS and decrease of antioxidant transcripts, which ultimately reduced in vitro developmental competence of bovine embryos. Based on these results, in vitro developmental competence of IVP bovine embryos was highly dependent on the coupled response between oxidative and ER stresses. These results increase our understanding of the mechanism(s) governing early embryonic development and may improve strategies for the generation of IVP embryos with high developmental competence.

VALIDAÇÃO DE UM PROGRAMA DE PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS COM TRANSPORTE DE OÓCITOS E DE EMBRIÕES POR LONGAS DISTÂNCIAS

This study aimed to evaluate the program of in vitro embryo production (IVEP) feasibility whose cattle oocyte maturation and embryo development occurred partly during the transport, fertilization (IVF) was performed with conventional or sexed semen and the embryos were transferred at different periods of development. Therefore, oocytes aspirated in the state of Bahia were transported to a commercial laboratory in São Paulo city, where the steps of in vitro production continued, the IVF was performed with sperm derived from conventional semen (CONV group) or sexed group (SEX group). On the sixth day of cultivation, the embryos produced were sent to Bahia and transferred to recipients at different periods of development (6.5, 7.0 and 7.5 days). The females had a mean of 46.18 ± 32.7 oocytes recovered, and 30.74 ± 24.3 viable oocytes by aspiration. The rates of embryos produced and pregnancy were 32.85% and 33.12%, respectively. The type of semen did not affect neither the number of cleaved oocytes nor the pregnancy rates, however, it affected the number of embryos produced, and the age of the embryo interfered in the pregnancy rates. The results demonstrated that the transport of oocytes and embryos was effective in cattle IVEP program, thus, it can be used in large scale.KEYWORDS: Bos indicus; sexed semen; transfer of embryos.

Cytokines, apoptosis and implantation

The follicle provides a unique micro-environment for the oocyte to develop and mature to a fertilizable gamete, within. Intrafollicular components, such as follicular fluid and cellular material are a potential resource for the noninvasive prediction of oocyte developmental potential. Using the cow as a model, we have established in vitro embryo production (IVP) methods that facilitate the individual tracking of oocytes and embryos, permitting a retrospective analysis of retained follicular components according to thedevelopmental outcomeof the oocyte during IVP. We have investigated the relationship between a range of parameters (including intrafollicular steroids and metabolomic profiles in follicle fluid and gene expression in follicular and cumulus cells) and oocyte development in vitro. Intrafollicular steroid concentrations in fluid from 3to 8-mm antral follicles were not predictive of oocyte developmental competence. However, metabolomic profiling of the same follicular fluid was highly predictive; several amino acids and polyunsaturated fatty acids positively correlated with blastocyst formation. Metabolomic analyses of follicular fluid from alternative bovine models of fertility have confirmed the predictive value of this approach. Employing both candidate and global gene expression analysis of transcript abundance in follicular and cumulus cells, we identified differentially expressed transcripts that were positively associated with blastocyst formation. However, many candidate genes are only predictivewithin themodel inwhich theywerefirst identified. Recently, we have focused on the role of progesterone (P4) during oocyte maturation. P4 has an anti-apoptotic effect on the cumulus–oocyte complex during maturation that is mediated via the nuclear P4 receptors. We have identified P4-responsive genes that have similar expression profiles in both the oocyte and their cumulus cells, implying their potential as biomarkers of oocyte quality. In particular, the pro-survival and cell cycle factor AVEN, whose dynamic regulation by P4 is reflected in both the oocyte and surrounding cumulus cells could potentially be exploited not just as an indicatorof oocytequality, but also as an indicator of IVCconditions. In summary, follicular components are an excellent resource for thenon-invasivepredictionofoocyte developmental potential; however, not all parameters are predictive across all models of oocyte development.

New device for the vitrification and in-straw warming of in vitro produced bovine embryos

Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1, 0.5 and 0M sucrose; protocol W3) or directly in 0.5M (protocol W1/0.5) or 0M sucrose (protocol W1/0). Similar survival rates were recorded 24h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5M sucrose) dilution/warming and different warming temperatures (45, 50, 60 and 70°C) were tested. When warming solution passed through the new vitrification/warming device at 45°C, 61.5% of blastocysts were fully re-expanded or hatched at 24h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45°C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions.

Confocal fluorescence assessment of bioenergy/redox status of dromedary camel (Camelus dromedarius) oocytes before and after in vitromaturation

BackgroundReproductive biotechnologies in dromedary camel (Camelus dromedarius) are less developed than in other livestock species. The in vitro maturation (IVM) technology is a fundamental step for in vitro embryo production (IVP), and its optimization could represent a way to increase the success rate of IVP. The aim of the present study was to investigate the bioenergy/oxidative status of dromedary camel oocytes before and after IVM by confocal microscopy 3D imaging.MethodsOocytes were retrieved by slicing ovaries collected at local slaughterhouses. Recovered oocytes were examined before and after IVM culture for nuclear chromatin configuration and bioenergy/oxidative status, expressed as mitochondria (mt) distribution and activity, intracellular Reactive Oxygen Species (ROS) levels and distribution and mt/ROS colocalization.ResultsThe mean recovery rate was 6 oocytes/ovary. After IVM, 61% of oocytes resumed meiosis and 36% reached the Metaphase II stage (MII). Oocyte bioenergy/redox confocal characterization revealed changes upon meiosis progression. Immature oocytes at the germinal vesicle (GV) stage were characterised by prevailing homogeneous mt distribution in small aggregates while MI and MII oocytes showed significantly higher rates of pericortical mt distribution organized in tubular networks (P < 0.05). Increased mt activity in MI (P < 0.001) and MII (P < 0.01) oocytes compared to GV stage oocytes was also observed. At any meiotic stage, homogeneous distribution of intracellular ROS was observed. Intracellular ROS levels also increased in MI (P < 0.01) and MII (P < 0.05) oocytes compared to GV stage oocytes. The mt/ROS colocalization signal increased in MI oocytes (P < 0.05).ConclusionsThis study provides indications that qualitative and quantitative indicators of bioenergy and oxidative status in dromedary camel oocytes are modified in relation with oocyte meiotic stage. These data may increase the knowledge of camel oocyte physiology, in order to enhance the efficiency of IVP procedures.

Assisted reproduction technologies in goats

Fertility in goats can be improved by natural methods (male effect and feeding) and by assisted reproductive technologies (ARTs). The main ARTs are: artificial insemination (AI), multiovulation and embryo transfer (MOET), in vitro embryo production (IVEP) and embryo cryopreservation. Nowadays, somatic cell nuclear transfer (SCNT) is the methodology to carry out reproductive cloning. Hormonal treatment to control and synchronize estrus and ovulation is an essential methodology to implementing the rest of the ARTs. In goats, AI, MOET and IVEP are used to increase goat reproductive efficiency, genetic improvement, transport genetic material and to preserve genetic resources for possible use in the future. Goats have been the principal domestic animal of choice for biomedical research especially for production of recombinant proteins secreted in milk. In recent years several studies have been carried out on transgenesis and cloned goats. However, ARTs need to be applied in healthy individuals to improve reproductive parameters. Poor animal health status, malnutrition and miss-management lie behind a reduction of fertility in spite of the techniques used.

Influence of Dominant Follicle and Corpus luteum on Recovery of Good Quality Oocytes for In vitro Embryo Production in Cattle

The quality of oocytes is more critical for the success of bovine in vitro embryo production (IVP) than the conditions of in vitro culture of these structures. Despite knowledge about bovine follicular development, especially gonadotropin-dependent phase, be considerably wide, knowledge about effects of the estrous cycle phase, follicular or luteal, on recovery Mini -review Article

Laparoscopic ovum pick up (LOPU) in goats: from hormonal treatment to oocyte possible destinations

Whether we are collecting oocytes from valuable donors as part of a program of in vitro embryo production (IVP) and/or to promote advances in emerging biotechnologies as cloning and transgenesis, the success rate depends on the development of methodological aspects of recovering oocytes. To succeed, sufficient number of good quality oocytes is the prerequisite for various reproductive techniques and laparoscopic ovum pick up (LOPU) is the recommended technique for obtaining them from live goats. However, the variability of the quantity and quality of the oocytes collected still limits the large-scale use of this technology. Under the current conditions, too large variability is reported with oocyte recovery rates ranging from 40 to 90%, and the number of harvested oocytes per female between 4 and 14, in different laboratories. This variability can depend on either intrinsic characteristic of the donors, such as breed, age or on aspects that we might be able to control, such as stimulation treatment, type of needle, aspiration pressure, among others. We believe that new investigations should contribute to significant improvement of LOPU yield. This review aims to report different factors influencing goat donor response for LOPU, presenting main steps for oocytes recovery as well as technical modifications proposed for improving LOPU efficiency. Furthermore, it is aimed to discuss about the potential use of goat oocytes after their recovery by LOPU and present overall results in goat IVP worldwide.

Evaluation of bovine (Bos indicus) ovarian potential for in vitro embryo production in the Adamawa plateau (Cameroon)

An abattoir study was conducted to evaluate the ovarian potential of 201 local zebu cattle from Ngaoundere, Adamawa region (Cameroon) for in vitro embryo production (IVEP). The ovaries were excised, submerged in normal saline solution (0.9%) and transported to the laboratory for a detailed evaluation. Follicles on each ovary were counted, their diameters (F) measured and were grouped into 3 categories: small (F &lt; 3 mm), medium (3 = F = 8 mm) and large (F &gt; 8 mm). Each ovary was then sliced into a petri dish; the oocytes were recovered in Dulbecco’s phosphate buffered saline, examined under a stereoscope (x10) and graded into four groups based on the morphology of cumulus oophorus cells and cytoplasmic changes of the oocytes. Grade I (GI): oocytes with more than 4 layers of bunch of compact cumulus cells mass with evenly granulated cytoplasm; grade II (GII): oocyte with at least 2–4 layers of compact cumulus cell mass with evenly granulated cytoplasm; grade III (GIII): oocyte with at least one layer of compact cumulus cell mass with evenly granulated cytoplasm; grade IV (GIV): denuded oocyte with no cumulus cells or incomplete layer of cumulus cell or expanded cells and having dark or unevenly granulated cytoplasm. The effects of both ovarian (ovarian localization, corpus luteum, size and weight of ovary) and non-ovarian factors (breed, age, body condition score (BCS) and pregnancy status of cow) on the follicular population and oocyte recovery rate were determined. There were an average of 16.75±0.83 follicles per ovary. The small, medium and large follicles were 8.39±0.60, 8.14±0.43 and 0.21±0.02 respectively. Oocyte recovery was 10.97±0.43 per ovary (65%). Oocytes graded I, II, III and IV were 3.53±0.19 (32.21%), 2.72±0.15 (24.82%), 2.24±0.15 (20.43%) and 2.47±0.20 (22.54%) respectively. The oocyte quality index was 2.26. Younger non pregnant cows having BCS of 3 and large ovaries presented higher number of follicles and oocyte quality (P &lt; 0.05) compared with other animals. Oocytes with quality (grade I and II) acceptable for IVEP constituted 57.15% of the harvest. This study indicated that factors such as age, pregnancy status, BCS and ovarian size must be taken into account to increase the potential of the ovary for IVEP.