Year-Long Phenotypical Study of Calves Derived From Different Assisted-Reproduction Technologies

Assisted reproductive technologies play a major role in the cattle industry. An increase in the use of in vitro-derived embryos is currently being seen around the globe. But the efficiency and quality of the in vitro-derived embryos are substandard when compared to the in vivo production. Different protocols have been designed to overcome this issue, one of those being the use of reproductive fluids as supplementation to embryo culture media. In this study, in vitro-derived calves produced with reproductive fluids added to their embryo production protocol were followed for the first year of life pairwise with their in vivo control, produced by artificial insemination (AI), and their in vitro control, produced with standard supplementation in embryo production. The objective was to assess if any differences could be found in terms of growth and development as well as hematological and biochemical analytes between the different systems. All the analysed variables (physical, hematological, and biochemical) were within physiological range and very similar between calves throughout the entire experiment. However, differences were more evident between calves derived from standard in vitro production and AI. We concluded that the use of reproductive fluids as a supplementation to the embryo culture media results in calves with closer growth and development patterns to those born by AI than the use of bovine serum albumin as supplementation.

Znanstvena istraživanja u 21. stoljeću - etički aspekti uporabe laboratorijskih životinja i alternativni modeli

U toksikološkim istraživanjima uz uporabu klasičnih (in vivo) istraživanja, primjenjuju se alternativni test sustavi. Korištenje laboratorijskih životinja, embrija, humanog i animalnog tkiva, kultura stanica i fetalnog seruma u istraživanjima smatra se etički problematičnim te se ograničava zakonima, pravilnicima i praksom. Razmatranjem načina kojima bi se neetičnost mogla izbjeći, došlo je do razvoja “3R” načela (akronim za tri pristupa koja bi se trebala provoditi pri istraživanjima na laboratorijskim životinjama), a to su: smanjenje/racionalizacija uporabe laboratorijskih životinja (engl. Reduction), načelo njihove zamjene (engl. Replacement) i poboljšanje uvjeta uzgoja, smještaja i skrbi za životinje (engl. Refinement). Većina je alternativnih testova toksičnosti još uvijek u postupku validacije. Pojedini in vitro testovi za istraživanja embriotoksičnosti (etički posebno osjetljivo područje) koja su priznala nadležna regulatorna tijela, su EST (engl. Embryonic Stem cell Test), WEC (engl. Whole- Embryo Culture) i MM (engl. MicroMass) test. Standardizacija protokola i uvođenje novih in vitro modela predstavlja važan segment napretka u toksikološkim istraživanjima. Znanstvena budućnost tu vidi mogućnost razvoja i implementacije načela etičnosti u istraživanja primjenjujući sustave koji će promišljeno i bez korištenja živih organizama dijelom nadomjestiti metode u biomedicini, veterinarskoj medicini, biotehnologiji i užem smislu - toksikologiji i farmakologiji.

Longitudinal Surface Measurements of Human Blastocysts Show That The Dynamics of Blastocoel Expansion Are Associated With Fertilization Method And Ongoing Pregnancy

Background: Despite all research efforts during this era of novel time-lapse morphokinetic parameters, a morphological grading system is still routinely being used for embryo selection at the blastocyst stage. The blastocyst expansion grade, as evaluated during morphological assessment, is associated with clinical pregnancy. However, this assessment is performed without taking the dynamics of blastocoel expansion into account. Here, we studied the dynamics of blastocoel expansion by comparing longitudinal blastocoel surface measurements using time-lapse embryo culture. Our aim was to first assess if this is impacted by fertilization method and second, to study if an association exists between these measurement and ongoing pregnancy. Methods: This was a retrospective cohort study including 225 couples undergoing 225 cycles of in vitro fertilization (IVF) treatment with time-lapse embryo culture. The fertilization method was either conventional IVF, intracytoplasmic sperm injection (ICSI) with ejaculated sperm or ICSI with sperm derived from testicular sperm extraction (TESE-ICSI). This resulted in 289 IVF embryos, 218 ICSI embryos and 259 TESE-ICSI embryos that reached at least the full blastocyst stage. Blastocoel surface measurements were performed on time-lapse images every hour, starting from full blastocyst formation (tB). Linear mixed model analysis was performed to study the association between blastocoel expansion, the calculated expansion rate (µm2/hour) and both fertilization method and ongoing pregnancy. Results: The blastocoel of both ICSI embryos and TESE-ICSI embryos was significantly smaller than the blastocoel of IVF embryos (beta -1121.6 µm2; 95% CI: -1606.1 to -637.1, beta -646.8 µm2; 95% CI: -1118.7 to 174.8, respectively). Still, the blastocoel of transferred embryos resulting in an ongoing pregnancy was significantly larger (beta 795.4 µm2; 95% CI: 15.4 to 1575.4) and expanded significantly faster (beta 100.9 µm2/hour; 95% CI: 5.7 to 196.2) than the blastocoel of transferred embryos that did not, regardless of the fertilization method. Conclusion: Longitudinal blastocyst surface measurements and expansion rates are promising non-invasive quantitative markers that can aid embryo selection for transfer and cryopreservation.

Embryo Culture, In Vitro Propagation, and Molecular Identification for Advanced Olive Breeding Programs

The high biodiversity of the olive tree is an important opportunity to develop sustainable plans to control Xylella fastidiosa (Xf) through breeding programs. Olive tree breeding activities have been limited due to various features of this species including the long time required for seed germination caused by the inhibition effect of the woody endocarp, the seed integument, and the endosperm. Starting from F1 seeds by cross-breeding, the embryo culture was compared with traditional seed germination, evaluating the effectiveness of in vitro multiplication of the plantlets for large-scale production. The isolated embryos were established on a new medium based on Rugini ‘84 macroelements, Murashige & Skoog ‘62 microelements, with Nitsch J. P. & Nitsch C. ‘69 vitamine and subcultured on Leva MSM modified. The results obtained confirmed that in vitro culture of olive embryos is a valid tool for increasing the percentage and speed of germination, helping to reduce the time of the olive breeding programs, offering the possibility to effectively propagate plantlets for further experiments.

Standardization of zygotic embryo culture from Nerium oleander L. and comparative analysis of biosynthesized cardiac glycosides within in vitro and acclimatized plants

The primary result of our experiment revealed that the germination percentage of N. oleander mature seeds is only 30%. From this observation, the concept of protocol standardization for zygotic embryo culture of this plant was originated. Zygotic embryo culture was proved an efficient in vitro multiplication system of N. oleander. The maximum germination percentage (96%) of zygotic embryos was observed on ¼ MS medium with 15 gm/L sucrose, whereas the best growth medium was optimized as ½ B5 with same sucrose concentration. The second part of this study was aimed to find out the cardiac glycoside accumulation pattern in both in vitro and acclimatized plants. For this purpose, one-month-old in vitro plantlets and acclimatized plants were subjected to LC-MS analysis and 09 cardiac glycosides were detected and quantified in both the systems. Most of the cardiac glycosides including odoroside A (32.71 mg/gm DW), odoroside H (4.69 mg/gm DW) and oleandrin (0.52 mg/gm DW) were found to be accumulated at maximum level within in vitro plantlets. CG 840b (1.89 mg/gm DW) is the only cardiac glycoside, which was maximally accumulated in acclimatized plants. From this study, it can be concluded that, zygotic embryo culture is a better choice for in vitro multiplication of N. oleander when compared to matured seeds and in vitro grown plantlets of this species favor cardiac glycosides biosynthesis in comparison to acclimatized plants. Therefore, all future research on the enrichment of cardiac glycosides from this plant may be conducted on zygotic embryos derived in vitro grown plantlets or cultures.

Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos

Background and Aim: Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos. Materials and Methods: In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M). Results: The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups. Conclusion: The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms.

Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubator

Conventional in vitro culture and manipulation of mouse embryos require a CO2 incubator, which not only increases the cost of performing experiments but also hampers the transport of embryos to the other laboratories. In this study, we established and tested a new CO2 incubator-free embryo culture system and transported embryos using this system. Using an Anaero pouch, which is a CO2 gas-generating agent, to increase the CO2 partial pressure of CZB medium to 4%–5%, 2-cell embryos were cultured to the blastocyst stage in a sealed tube without a CO2 incubator at 37°C. Further, the developmental rate to blastocyst and full-term development after embryo transfer were comparable with those of usual culture method using a CO2 incubator (blastocyst rate: 97% versus 95%, respectively; offspring rate: 30% versus 35%, respectively). Furthermore, using a thermal bottle, embryos were reliably cultured using this system for up to 2 days at room temperature, and live offspring were obtained from embryos transported in this simple and very low-cost manner without reducing the offspring rate (thermal bottle: 26.2% versus CO2 incubator: 34.3%). This study demonstrates that CO2 incubators are not essential for embryo culture and transportation and that this system provides a useful, low-cost alternative for mouse embryo culture and manipulation.

Reproductive Characteristics of the Selected Cocoa (Theobroma cacao L.) Clones after Regenerated from the Somatic Embryogenesis Culture

This study was conducted to evaluate the reproductive characteristics of 4 elite cocoa clones (MCBC1, PBC230, KKM22 and KKM4) propagated via somatic embryogenesis culture. From the findings, all clones have similar reproductive characteristics with clones from conventional grafted. However, only KKM4 clone from immature zygotic embryo culture produced the shortest staminode to style distance of 1.83 mm. This consequently influenced flower stability by reducing the efficiency of pollination by insects. It was found that this clone also has the highest number of flowers drop after anthesis (5 flowers) and lowest production of cherelle (5 cherelles). Further observation revealed that floral development from first bud visible (BBCH51) to flower anthesis (BBCH68) of all clones took around 31 days. These cocoa flowers which remained receptive soon after anthesis at 10 am (day-31) until the next day (day-32) suggesting 2 days’ period of receptivity. HIGHLIGHTS It is crucial to assess the presence of off-type characteristics in the reproductive organ structure such as the distance between staminode to style, period of reproductive cycle and stigmatic receptivity of cocoa clones regenerated from somatic embryogenesis The converging and parallel type of staminode to style distances are the ideal flower spatial arrangements for the optimal pollination in cocoa plant compared to splay type Only KKM4 clone propagated from immature zygotic embryo culture showed variation in the distance between staminode to style distance and this caused pollination failure by insect which then consequently caused minimum cherelle production All regenerated cocoa clones observed with typical period of the reproductive cycle and stigmatic receptivity GRAPHICAL ABSTRACT