Linoleic acid (LA; 18:2 n-6) is the most abundant fatty acid in bovine follicular fluid, and it was previously reported that LA concentration significantly decreases when follicle size increases. This suggests that LA may have a role in the regulation of oocyte maturation. The present study investigated the effect of LA supplementation on bovine oocyte maturation and early embryo development in vitro. Treatment of cumulus-oocyte complexes (COCs) with LA significantly inhibited cumulus cell expansion and retarded development of the oocytes to the metaphase II (MII) stage in a dose-dependent manner. This effect was reversible, and the oocytes developed to the MII stage after extended culture in the absence of LA. Treatment of COCs with LA also resulted in a significantly lower percentage of cleaved embryos and blastocyst yield. Furthermore, COCs treated with LA had significant effects compared with controls in i) increasing prostaglandin E(2) concentration in the medium, ii) decreasing intracellular cAMP at 6 and 24 h of maturation and iii) decreasing phosphorylation of the MAPK1 and 3 at 24 h, and AKT at 6 h of maturation. In conclusion, LA supplementation to bovine oocytes during maturation altered the molecular mechanisms regulating oocyte maturation and resulted in decreased percentage of oocytes at MII stage and inhibition of the subsequent early embryo development. These data provide evidence for adverse effects of LA on oocyte development, which can be associated with dietary increased level of LA in the follicular fluid and the decline in fertility in farm animals and human.

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Impact of linoleic acid on bovine oocyte maturation and embryo development.

Melatonin has a wide variety of beneficial actions at the level of the gonads and their adnexa. Some actions are mediated via its classic membrane melatonin receptors while others seem to be receptor-independent. This review summarizes many of the published reports which confirm that melatonin, which is produced in the ovary, aids in advancing follicular maturation and preserving the integrity of the ovum prior to and at the time of ovulation. Likewise, when ova are collected for in vitro fertilization-embryo transfer, treating them with melatonin improves implantation and pregnancy rates. Melatonin synthesis as well as its receptors have also been identified in the placenta. In this organ, melatonin seems to be of particular importance for the maintenance of the optimal turnover of cells in the villous trophoblast via its ability to regulate apoptosis. For male gametes, melatonin has also proven useful in protecting them from oxidative damage and preserving their viability. Incubation of ejaculated animal sperm improves their motility and prolongs their viability. For human sperm as well, melatonin is also a valuable agent for protecting them from free radical damage. In general, the direct actions of melatonin on the gonads and adnexa of mammals indicate it is an important agent for maintaining optimal reproductive physiology.

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Peripheral reproductive organ health and melatonin: ready for prime time.

SUMMARY Melatonin is a multifunctional molecule that mediates several circadian and seasonal reproductive processes. The exact role of melatonin in modulating reproduction, however, is not fully understood—especially its effects on the ovarian follicles and oocytes. This study was conducted to investigate the expressions of the ASMT and melatonin-receptor MTNR1A and MTNR1B genes in bovine oocytes and their cumulus cells, as well as the effects of melatonin on oocyte nuclear and cytoplasmic maturation in vitro. Cumulus-oocyte complexes (COCs) from abattoir ovaries were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10, 50, and 100 ng/ml. The expression of ASMT, MTNR1A, and MTNR1B genes was evaluated by RT-PCR. Moreover, the effects of melatonin on cumulus cell expansion, nuclear maturation, mitochondrial characteristics and COCs steroidogenesis were investigated. Furthermore, the level of reactive oxygen species (ROS) was evaluated in denuded oocytes. Our study revealed that ASMT and MTNR1A genes were expressed in COCs, while the MTNR1B gene was expressed only in oocytes. Additionally, melatonin supplementation at 10 and 50 ng/ml to in vitro maturation medium significantly enhanced oocyte nuclear maturation, cumulus cell expansion and altered the mitochondrial distribution patterns, but had no effects on oocyte mitochondrial activity and COCs steroidogenesis. Melatonin-treated oocytes had a significantly lower level of ROS than controls. The presence of melatonin receptors in COCs and its promoting effects on oocyte nuclear and cytoplasmic events, indicate the potentially important roles of this hormone in regulating bovine oocyte maturation. Moreover, the presence of ASMT transcript in COCs suggests the possible involvement of these cells in melatonin biosynthesis.

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Evidence of melatonin synthesis in the cumulus oocyte complexes and its role in enhancing oocyte maturation in vitro in cattle

Fusarium mycotoxins: Effects on reproductive function in domestic animals—A review

Effects of follicular atresia and size on the developmental competence of bovine oocytes: A study using the well-in-drop culture system

The objective of the present study was to investigate the correlation between the degree of cumulus expansion and in vitro development of porcine cumulus-oocytes complexes (COCs) matured and fertilized in vitro. The COCs were matured in the maturation medium (IVMM) supplemented with 15% or 5% of porcine follicular fluid (PFF) from small, medium and large follicles ( 5 mm, respectively). COCs cultured in IVMM with PFF for 48 h displayed less expansion than those cultured in IVMM alone (P 0.05). After in vitro fertilization, the oocytes failed to develop to the morula/blastocyst stages except for those matured in IVMM supplemented with 15% or 5% PFF obtained from >5 mm follicles for the first 24 h and followed by in IVMM alone for the second 24 h (12.5% and 11.1% of the embryos developed to morulae and blastocysts, respectively). The expanded cumulus areas of COCs were significantly positively correlated with their in vitro development (p=0.0058, 0.0001 and 0.0348 for the percentages of embryos developed to 2-4 cell, beyond 4 cell and morula and blastocyst stages, respectively). In conclusion, PFF had an inhibiting effect on cumulus expansion, and the inhibitory effect decreased progressively with the increase in size of follicles from which PFF was obtained, and the action of PFF on cumulus expansion was affected by the PFF culture time. The areas of the expanded cumulus mass may be used as a parameter to predict development of porcine oocytes matured and fertilized in vitro.

Predictive value of the area of expanded cumulus mass on development of porcine oocytes matured and fertilized in vitro.

The aim of the present experiment was to investigate the effect of follicular size on oocyte cumulus-expansion, in vitro fertilization and subsequent developmental competence. After maturation culture, the rates of porcine oocytes with expanded cumulus derived from antral follicles ≥5 mm, 4-4.9 mm, 3-3.9 mm and 2-2.9 mm in diameter were 90.5%, 89.7%, 85.4% and 67.4%, respectively. After in vitro fertilization, the developmental competence of oocytes dependently increased with follicular size. Higher cleavage rates and higher proportions of 3-4-cell embryos were obtained from ≥4 mm follicles compared to 2-2.9 mm follicles (P<0.05). Although the proportions of 6-8-cell and 12-16-cell embryos obtained from ≥5 mm, 4-4.9 mm, 3-3.9 mm and 2-2.9 mm follicles showed no significant differences, embryos obtained from 2-2.9 mm follicles showed a complete failure to develop beyond the 8-cell stage, embryos obtained from 3-3.9 mm follicles failed to develop beyond the 16-cell stage and none of embryos developed beyond the early stage of morula. The percentages of expanded cumulus-oocyte complexes (COCs) and 2-cell cleavage rates of oocytes derived from 2-2.9 mm follicles were not significantly different when the oocytes were matured for 36 h , 42 h and 48 h in vitro. The exposure to 5% or 15% porcine follicular fluid (PFF) from the large follicles during in vitro maturation (IVM) had no significant effect on the cleavage and subsequent developmental rates of porcine oocytes compared to PFF from small follicles.

Effect of Follicle Size on Cumulus-Expansion, In Vitro Fertilization and Development of Porcine Follicular Oocytes

Purpose: The present study was to investigate the impact of type and state of co-culture cells on developmental competence of porcine oocytes matured and fertilized in vitro. Methods: Porcine zygotes were co-cultured with granulosa cells (GCs) (Group 1) or porcine oviductal epithelial cells (pOECs) at follicular stage (Group 2), ovulation stage (Group 3) or corpus luteum (CL) stage (Group 4) or cultured in a medium without co-culture cells (control group). Results: The proportion of oocytes developed to 2-cell stage embryos in Group 2 was similar to that in control group, but significantly (p < 0.05) lower than that in Groups 1, 3 and 4. The proportions of oocytes developed to ≥4-cell stage embryos in Groups 3 and 4 were significantly (p < 0.05) higher than that in Groups 1 and 2. At 144 h after insemination, 12.0, 14.8 and 20.0% of oocytes developed to blastocysts in Groups 1, 3 and 4, respectively. However, no embryos in control group developed beyond 4-cell stage and no embryos in Group 2 developed to blastocyst stage. Conclusion: As compared with GCs and pOECs at follicular stage, the pOECs at ovulation and CL stages had a better competence to support porcine embryo development under in vitro conditions.

Jia Tong Ding

Papers

Predictive value of the area of expanded cumulus mass on development of porcine oocytes matured and fertilized in vitro.

Effect of Follicle Size on Cumulus-Expansion, In Vitro Fertilization and Development of Porcine Follicular Oocytes

Effects of type and state of co-culture cells on in-vitro development of porcine oocytes matured and fertilized in vitro.