Characterization of the Germplasm Bank for the Spanish Autochthonous Bull Breed "Asturiana de la Montaña".

Semen cryobanks are critical for preserving autochthonous and rare breeds. Since sperm cryopreservation has been optimized for commercial breeds, non-commercial ones (often endangered) must be characterized to ensure the germplasm's viability. This study reports an investigation of the "Asturiana de la Montaña" breed (AM), a valuable Spanish autochthonous cattle breed adapted to the mountainous Atlantic environment. The survey included cryopreserved semen doses from 40 bulls stored at the Principado de Asturias Germplasm Bank. Data were obtained from the routine fresh semen analysis, CASA (motility), and flow cytometry analyses of fresh and post-thawing semen, and the 56-day non-return-rate (NRR) in heifers and cows (all results as 1st and 3rd quartiles). Fresh samples (artificial vagina) were within the normal range for cattle (4-6 mL, 5-10 × 109/mL; mass motility 5). Post-thawing results showed motility below typical for commercial breeds (total motility 26-43%, progressive 14-28%), with higher values for viability (47-62%). Insemination results showed a good performance for this breed (NRR: 47-56%; higher for heifers). Sperm volume increased with age, with little or no effects on sperm quality. Few associations were found between post-thawing quality or freezability and NRR, LIN being the variable more strongly associated (positively). The AM semen bank shows a good prospect for preserving and disseminating the genetics of this breed. This survey indicates that dedicated research is needed to adapt freezing protocols to this breed, optimizing post-thawing results.

Use of native chicken breeds (Gallus gallus domesticus) for the development of suitable methods of Cantabrian capercaillie (Tetrao urogallus cantabricus) semen cryopreservation.

BACKGROUND: The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals. OBJECTIVES: In this study, we analyzed the response of capercaillie sperm to the freezing-thawing process for contributing to the development of a semen cryobank of Cantabrian capercaillie. METHODS: We used domestic chicken as the animal model in order to obtain the freezing protocol before applying on capercaillie. In the first experiment, two different extenders (EK and LR84) and different concentrations [4% and 6% dimethyl-acetamide (DMA) v:v] of cryoprotectants were evaluated using in-straw freezing method in domestic chickens. A pilot study in capercaillie males, using the same conditions evaluated in chicken, was performed. RESULTS: In chicken, we found that the LR84-4% DMA media provided the best results for freezing semen. In capercaillie study, LR84 extender seemed to be the most appropriate diluent and 4% was the better dose of DMA cryoprotectant agent. Further, based on previous studies carried out in rooster samples, we also tested the glycerol (8% v/v) as a cryoprotectant for capercaillie semen cryopreservation. CONCLUSIONS: Our results suggest that sperm from both domestic and wild species had a similar response to freezing-thawing processes. Mediterranean chickens may be used as a suitable model for developing sperm freezing protocols that can be extrapolated to threatened capercaillie populations. In addition, LR84 media with glycerol was the most efficient extender to freeze capercaillie sperm native.

Post-Thaw Sperm Quality and Functionality in the Autochthonous Pig Breed Gochu Asturcelta.

Genetic resource banks (GRB) preserve the genetic material of endangered, valuable individuals or genetically relevant breeds. Semen cryopreservation is a crucial technique to reach these goals. Thus, we aimed to assess the sperm parameters of semen doses from the native pig breed Gochu Asturcelta stored at the GRB of Principado de Asturias (GRB-PA, Gijón, Spain), focusing on intrinsic and extrinsic (boar, season) factors. Two straws per boar (n = 18, 8-71 months of age) were thawed, pooled, and assessed after 30 and 150 min at 37 °C by CASA (computer-assisted sperm analysis system; motility and kinematic parameters) and flow cytometry (viability, acrosomal status, mitochondrial activity, apoptosis, reactive oxygen species, and chromatin status). The effects of age, incubation, and season on post-thawing quality were determined using linear mixed-effects models. Parameters were on the range for commercial boar breeds, with chromatin status (SCSA: fragmentation and immaturity) being excellent. Incubation decreased sperm quality and functionality. The boar age did not have a significant effect (p > 0.05), but the between-boar variability was significant (p < 0.001). The season significantly affected many parameters (motility, kinematics, viability, acrosomal status, mitochondrial activity), especially after 150 min of incubation. In general, samples collected in spring and summer showed higher quality post-thawing, the lowest in winter. In conclusion, the sperm doses from the Gochu Asturcelta breed stored at the GRB-PA showed excellent chromatin status and acceptable characteristics after thawing. Therefore, boar and seasonal variability in this autochthonous breed could be relevant for cryobank management.

Assessment of a germplasm bank for the autochthonous cattle breed Asturiana de la Montaña: Extender (Biociphos vs. BIOXCell) affected sperm quality but not field fertility.

Semen banking is critical to preserving rare and autochthonous breeds. However, protocols can change with time, leaving heterogeneous semen batches. The objective of this study was to assess differences in sperm quality and field fertility. We report differences between batches frozen with the Biociphos and BIOXCell extenders in the Asturiana de la Montaña cryobank (autochthonous and endangered breed, Northern Spain). Doses from 48 bulls were analysed by CASA and flow cytometry. The 85-days non-return rates from AI records were used to assess the fertility of 23,853 AI. BIOXCell showed higher quality post-thawing. Differences increased after a 5-hr incubation at 37°C, and Biociphos yielded doses with lower resilience. Field fertility did not differ between extenders (Biociphos: 57.4% ± 1.2; BIOXCell: 56.6% ± 3.0), possibly because of AI protocols compensating for differences in quality. However, this needs to be confirmed by controlled intervention studies. In conclusion, batches frozen with Biociphos may require specific strategies for compensating for the lower sperm quality. Regular surveys and evaluation of cryobank procedures may be useful to characterizing stored batches and defining strategies to guaranteeing success in their future use.

Expression and localization of interleukin 1 beta and interleukin 1 receptor (type I) in the bovine endometrium and embryo.

The interleukin-1 (IL1) system likely mediates mammalian embryo-maternal communication. In cattle, we have reported that the uterine fluid of heifers carrying early embryos shows downregulated IL1 beta (IL1B), which could lead to reduced NFkB expression and dampening of maternal innate immune responses. In this work, we assessed the expression of IL 1 beta (IL1B) and its receptor, interleukin 1 receptor type I (IL1R1) in the bovine endometrium and embryos by RT-PCR, immunohistochemistry and Western blot at the time of blastocyst development. Day 8 endometrium, both collected from animals after transfer of day 5 embryos (ET) and sham transferred (ST), showed IL1B and IL1R1 mRNA transcription and protein co-localization. Similarly, day 8 blastocyst, from ET animals and entirely produced in vitro, showed IL1R1 mRNA transcription and IL1B and IL1R1 protein co-localization. IL1B mRNA was detected in the analyzed blastocysts, but at very low levels that precluded its quantification. IL1B and IL1R1 immunostaining was observed in luminal epithelial cells, glandular epithelium and stromal cells. The presence of embryos increased endometrial IL1B protein locally, while no differences regarding IL1R1 protein and IL1B and IL1R1 mRNA were detected. These results suggest that the early preimplantation bovine embryo in the maternal tract might interact with the maternal immune system through the IL1 system. Such a mechanism may allow the embryo to elicit local endometrial responses at early stages, which are required for the development of a receptive endometrium.

Metabolomic prediction of pregnancy viability in superovulated cattle embryos and recipients with fourier transform infrared spectroscopy.

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared spectroscopy (FTIR) metabolomics to identify spectral models predictive of pregnancy outcome. Embryos collected on Day 6 from superovulated cows in 2 countries were individually cultured in synthetic oviduct fluid medium with BSA for 24 h before embryo transfer. Spent CM, blank controls, and plasma samples (Day 0 and Day 7) were evaluated using FTIR. The spectra obtained were analyzed. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the ROC curve (AUC). Endpoints considered were Day 60 pregnancy and birth. High AUC was obtained for Day 60 pregnancy in CM within individual laboratories (France AUC = 0.751 ± 0.039, Spain AUC = 0.718 ± 0.024), while cumulative data decreased the AUC (AUC = 0.604 ± 0.029). Predictions for CM at birth were lower than Day 60 pregnancy. Predictions with plasma at birth improved cumulative over individual results (Day 0: France AUC = 0.690 ± 0.044; Spain AUC < 0.55; cumulative AUC = 0.747 ± 0.032). Plasma generally predicted pregnancy and birth better than CM. These first results show that FTIR metabolomics could allow the identification of embryos and recipients with improved pregnancy viability, which may contribute to increasing the efficiency of selection schemes based on ET.

Efficient derivation of bovine embryonic stem cells needs more than active core pluripotency factors.

Pluripotency can be captured in vitro, providing that the culture environment meets the requirements that avoid differentiation while stimulating self-renewal. From studies in the mouse embryo, two kinds of pluripotent stem cells have been obtained from the early and late epiblast, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), representing the naive and primed states, respectively. All attempts to derive convincing ESCs in ungulates have been unsuccessful, although all attempts were based on the assumption that the conditions used to derive mouse ESCs or human ESC could be applied in other species. Pluripotent cells derived in primates, rabbit, and pig strongly indicate that the state of pluripotency of these cells is, in fact, closer to EpiSCs than to ESCs, and thus depend on fibroblast growth factor (FGF) and Activin signaling pathways. Based on this observation, we have tried to derive EpiSC from the epiblast of bovine elongated embryos as well as ESCs from Day-8 blastocysts. We here show that the core transcription factors Oct4/Sox2/Nanog can be used as markers of pluripotency in the bovine since their expression was restricted to the developing epiblast after Day 8, and disappeared following differentiation of both the ESC-like and EpiSC-like cultures. Although FGF and Activin pathways are indeed present and active in the bovine, it is not sufficient/enough to maintain a long-term pluripotency ex vivo, as was reported for mouse and pig EpiSCs.

Gene expression in early expanded parthenogenetic and in vitro fertilized bovine blastocysts.

Mammalian oocytes can undergo artificial parthenogenesis in vitro and develop to the blastocyst stage. In this study, using real-time PCR, we analyzed the expression of genes representative of essential events in development. In vitro matured oocytes were either fertilized or activated with ionomycin + 6-DMAP and cultured in simple medium. The pluripotency-related gene Oct3/4 was downregulated in parthenotes, while the de novo methylation DNMT3A gene was unchanged. Among the pregnancy recognition genes, IFN-t was upregulated, PGRMC1 was downregulated and PLAC8 was unchanged in parthenotes. Among the metabolism genes, SLC2A1 was downregulated, while AKR1B1, COX2, H6PD and TXN were upregulated in parthenotes; there was no difference in SLC2A5. Among the genes involved in compaction/blastulation, GJA1 expression increased in parthenotes, but no differences were detected within ATP1A1 and CDH1. Expression of p66(shc) and the Bax/Bcl2 ratio were higher in parthenotes, and there was no difference in p53. Parthenotes and embryos may differ in the way they stimulate apoptosis, with a preponderant role for p66(shc) within parthenotes. Differentially affected functions may also include pluripotency, de novo methylation and early embryonic signalling.

Changes in testosterone or temperature during the in vitro oocyte culture do not alter the sex ratio of bovine embryos.

High follicular testosterone levels have been associated with a skew in the sex ratio in favor of males following in vitro fertilization, whereas egg incubation temperature has been found to influence sex ratio in some reptiles. The incubation temperature interferes with the aromatase activity, resulting in a sex determination mechanism thought to be lost in mammals. In this work we aimed to test the effects of testosterone on sex ratio of bovine embryos produced in vitro and to determine whether effects of sex and temperature are effectively decoupled in mammals. Bovine oocytes were in vitro matured for 22 hr in TCM199, PVA, FSH and LH after a 22 hr meiotic arrest in TCM199, PVA and roscovitine 25 microM. Matured oocytes were in vitro fertilized and cultured up to Day 3, and embryos having three or more cells were sexed. In the first experiment, testosterone (0, 30, 300 and 1,500 nM), present both during meiotic inhibition and subsequent in vitro maturation (IVM), did not affect development rates or embryonic sex ratio. In the second experiment, increasing incubation temperatures (38, 39 or 40 degrees C) during meiotic inhibition and subsequent IVM, reduced embryo development, but did not change the sex ratio. Under our experimental conditions, testosterone does not promote a preferential selection of Y-chromosome bearing spermatozoa by the oocyte, and temperature and sex ratio seems to be decoupled in mammals.

Biological differences between in vitro produced bovine embryos and parthenotes.

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66(shc) and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1. Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66(shc) and p53 respectively, and in their mechanisms to control pluripotency and de novo methylation.

Development and quality of bovine morulae cultured in serum-free medium with specific retinoid receptor agonists.

Retinoids regulate development and differentiation of the bovine blastocyst in vitro, although the underlying mechanisms remain to be clarified. A challenge in reproductive biotechnology is the identification of pathways that regulate early embryonic development and their influence on blastocyst differentiation, apoptosis and survival to cryopreservation as traits of embryo quality. The present paper analyses the effects of short-term exposure (24 h) to retinoids on in vitro-produced bovine morulae. Immature cumulus oocyte complexes were in vitro matured and fertilised. Presumptive zygotes were subsequently cultured in modified synthetic oviduct fluid up to Day 6, in which morulae were randomly allocated to the different experimental groups. The treatments consisted of 0.1 microM LG100268 (LG; a retinoid X receptor agonist), 0.7 microM all-trans retinoic acid (ATRA; a retinoic acid receptor agonist) or no additives. Day 8 blastocyst development was increased in the ATRA-treated group compared with the LG and untreated embryos. In Day 7 embryos, the number of total cells and cells allocated to the trophectoderm were higher in the ATRA-treated group compared with untreated embryos. Apoptosis in the inner cell mass increased after LG treatment, whereas ATRA had no effect. After vitrification and warming, survival and hatching rates of Day 7 blastocysts did not change with retinoid treatment. Within the LG-treated and untreated blastocyst groups, survival and hatching rates were higher for Day 7 than Day 8 embryos; however, Day 8 blastocysts treated with ATRA showed improved hatching rates. In conclusion, treatment of morulae with ATRA in serum-free medium improves embryo development and quality without increasing the incidence of apoptosis and necrosis.

Expression and proteasomal degradation of the major vault protein (MVP) in mammalian oocytes and zygotes.

Major vault protein (MVP), also called lung resistance-related protein is a ribonucleoprotein comprising a major part (>70%) of the vault particle. The function of vault particle is not known, although it appears to be involved in multi-drug resistance and cellular signaling. Here we show that MVP is expressed in mammalian, porcine, and human ova and in the porcine preimplantation embryo. MVP was identified by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) peptide sequencing and Western blotting as a protein accumulating in porcine zygotes cultured in the presence of specific proteasomal inhibitor MG132. MVP also accumulated in poor-quality human oocytes donated by infertile couples and porcine embryos that failed to develop normally after in vitro fertilization or somatic cell nuclear transfer. Normal porcine oocytes and embryos at various stages of preimplantation development showed mostly cytoplasmic labeling, with increased accumulation of vault particles around large cytoplasmic lipid inclusions and membrane vesicles. Occasionally, MVP was associated with the nuclear envelope and nucleolus precursor bodies. Nucleotide sequences with a high degree of homology to human MVP gene sequence were identified in porcine oocyte and endometrial cell cDNA libraries. We interpret these data as the evidence for the expression and ubiquitin-proteasome-dependent turnover of MVP in the mammalian ovum. Similar to carcinoma cells, MVP could fulfill a cell-protecting function during early embryonic development.

Proteasomal interference prevents zona pellucida penetration and fertilization in mammals.

The ubiquitin-proteasome pathway has been implicated in the penetration of ascidian vitelline envelope by the fertilizing spermatozoon (Sawada et al., Proc Natl Acad Sci U S A 2002; 99:1223-1228). The present study provides experimental evidence demonstrating proteasome involvement in the penetration of mammalian zona pellucida (ZP). Using porcine in vitro fertilization as a model, penetration of ZP was completely inhibited by specific proteasomal inhibitors MG-132 and lactacystin. Three commercial rabbit sera recognizing 20S proteasomal core subunits beta-1i, beta-2i, alpha-6, and beta-5 completely blocked fertilization at a very low concentration (i.e., diluted 1/2000 to 1/8000 in fertilization medium). Neither proteasome inhibitors nor antibodies had any effects on sperm-ZP binding and acrosome exocytosis in zona-enclosed oocytes or on fertilization rates in zona-free oocytes, which were highly polyspermic. Consistent with a possible role of ubiquitin-proteasome pathway in ZP penetration, ubiquitin and various alpha and beta type proteasomal subunits were detected in boar sperm acrosome by specific antibodies, immunoprecipitated and microsequenced by MALDI-TOF from boar sperm extracts. Antiubiquitin-immunoreactive substrates were detected on the outer face of ZP by epifluorescence microscopy. This study therefore provides strong evidence implicating the ubiquitin-proteasome pathway in mammalian fertilization and zona penetration. This finding opens a new line of acrosome/ZP research because further studies of the sperm acrosomal proteasome can provide new tools for the management of polyspermia during in vitro fertilization and identify new targets for contraceptive development.