The prepubertal sheep ovaries have a large number of small follicles containing oocytes with a capability of developing to upper stages of nuclear maturation, cleaving subsequent post insemination and finally being a blastocyst, that can be transferred to surrogate ewes for progressing of animal breeding programs. Whereas, it has shown that oocytes from prepubertal animals have low competence to develop to higher stages. Accordingly, in this study we have tried to do experiments to improve this deficiency by using Alpha linolenic acid (ALA) and Linoleic Acid (LA). Also, we have tried to improve survival of sheep embryos cultured in vitro using Hyaluronan (HA). Polyunsaturated fatty acids (PUFAs) have shown to have beneficial effects on oocyte maturation and embryo development in vivo and in vitro conditions. Also, PUFAs constitute the major portion of the fatty acid content of the follicular fluid in small and large follicles. ALA and LA from PUFAs used in the present study in 2 separate experiments (experiment 1 for ALA and experiment 2 for LA). To our knowledge there are no previous reports using ALA and LA in prepubertal sheep oocytes in vitro. We used in three concentrations (50, 100 and 200µM) of ALA and LA in maturation media. Subsequently, we evaluated parameters such as cumulus cell expansion, nuclear maturation, secretion of prostaglandins (PGE2 and PGF2α) and steroids (E2 and P4), two pro-nuclei, polyspermy, asynchrony, cleavage, blastocyst rate and embryo quality via counting total cell number and apoptotic cells. In experiment 1, oocytes nuclear maturation and the number of fully expanded cumulus cells were reduced in 200 µM ALA treatment compared to other groups (P≤0.05). Supplementation with ALA increased both PGE2 and PGF2α concentration in the spent media (P≤0.05). No differences were observed in blastocyst development among control (12.2%) and 50, 100 and 200 µM ALA groups (6.9, 11.5 and 14.0%, respectively). However, total cells (46.50±5.85, 67.94±6.71, 45.20±6.37, and 59.80±5.51, respectively, P≤0.05) and apoptotic cell number (6.45±0.89, 2.48±0.81, 4.02±1.15, and 3.67±1.15, respectively, P≤0.05) were significantly improved. After IVM, E2 concentration was lower and P4 concentration was higher in ALA groups compared to control (P≤0.05). In conclusion, these results showed that ALA affects prepubertal sheep embryo quality associated with alteration of releasing reproductive hormones. In experiment 2, no changes were observed in the number of oocytes achieving MII nuclear maturation (91.8, 91.6, 87.9, 93.1 and 93.1, respectively). Production of PGE2 and PGF2α increased in all LA concentrations compared to control (P≤0.05). The ratio of PGE2/PGF2α was not altered. LA at 50µM significantly improved the rate of 2 pro-nuclear (2PN) compared to control (57.89 vs. 45.45, respectively, P≤0.05). There were no differences in cleaved embryos and blastocyst rates. However, embryo quality was improved by 50µM of LA with increase in total cell numbers compared to control (63.88±4.54 vs. 53.35±3.64, P≤0.05, respectively). There was no difference in apoptotic cell numbers. Also, production of E2 decreased significantly while there were not differences in P4 production and the rate of E2/P4 ratio. In conclusion, LA supplementation to prepubertal sheep oocytes in IVM media negatively altered the fully expanded cumulus cells significantly without inhibition of MII nuclear stage percentage of oocytes. The results from the present study provide evidence in increased number of zygotes with normal 2PN and also showed beneficial effects of low level LA on embryo quality of blastocysts at 8 day of post insemination in serum free media. HA is a polysaccharide with long polymer chains of sugars and has found in the extracellular matrix and intercellular matrix of animal tissues. HA is found enormously as a plentiful Glycosaminoglycan (GAG) in the female reproductive tract like uterus, oviduct and follicular fluids. Furthermore, it has been illustrated that HA plays a role in introducing a delay of death in the oocyte with preventing of oocyte from fragmentation in porcine. Also HA improves in vitro produced bovine embryos to develop to the blastocyst stage. It has also been indicated that embryo cryo-survival improvement is due to the HA added to cryopreservation medium HA numerically increased blastocyst percentage at 7-day (33±5.7, 32±6.0, 35±5.5; P≥0.05) and survival rates 48 h after culture in serum free media (63±17.1, 83±15.2, 58±14.2; P≥0.05) as compared to the respective controls (25±5.2, 38±17.1). It increased the total cell (TC) number (83.6±4.6, 100.7±3.8, 97.2±3.7, 105.0±3.9; P<0.05) and trophectoderm cells (TE) (58.4±3.8, 74.2±3.2, 75.6±3.3, 80.1±3.4; P<0.05) at 7-day embryos. Survived embryos had higher TC (63.2±3.7, 130.8±3.6, 113.9±5.2, 149.8±5.4; P<0.05), TE (42.9±3.0, 96.7±3.1, 85.2±4.5, 111.9±4.7; P<0.05) and ICM (20.3±2.2, 32.9±1.8, 27.7±2.6, 36.5±2.7; P<0.05). The results indicate that HA improves the embryo development and viability even quality which might have implication for improving embryo transfer.
|Date of Award||28 Nov 2014|
- Universitat Autònoma de Barcelona (UAB)
|Supervisor||M. Teresa Paramio Nieto (Director) & Ali Fouladi-Nashta (Director)|