The effects of melatonin on in vitro oocyte competence and embryo development in sheep

The aim of this study was to assess the effects of melatonin on the in vitro maturation and fertilization of sheep oocytes, and on the in vitro culture of the embryos. Oocytes from sheep ovaries collected at the slaughterhouse were divided into four groups, two of which were treated with either 10 M (M5) or 10 M (M6) melatonin, while the other two groups served as untreated controls (C5 and C6). After in vitro fertilization with fresh ram semen, the embryos produced in each group were divided into two sets, one cultured with melatonin (M5M, C5M, M6M and C6M), and the other without melatonin (M5C, C5C, M6C, and C6C). A melatonin concentration of 10 M increased maturation rate (82.5% vs. 73.7% for M6 and C6, respectively; P < 0.05) and tended to improve cleavage rate 36 hours after in vitro fertilization (79.4% vs. 72.6% for M6 and C6, respectively, P = 0.08). A higher melatonin concentration (10 M) did not have significant effects on those parameters. Blastocyst rates on day 8 did not differ significantly among groups. Additional key words: in vitro fertilization, in vitro maturation.


Introduction
The use of melatonin implants to improve reproduction in sheep is widely spread in some Mediterranean countries.In the last few years some reports have revealed that melatonin not only modifies the perception of photoperiod by ewes at hypothalamic level but also can exert its influence at other targets of the reproductive system (Abecia et al., 2008).Melatonin, an endogenous hormone produced by several organs,
particularly the pineal gland (Huether, 1993), is essential for sleep and temperature regulation, circadian and seasonal rhythmic cycles, and reproductive physiology (Arendt, 1998;Arendt, 2003).Melatonin is used widely to advance the breeding season in sheep and is an effective method of inducing oestrous cycles, increasing lambing rates and prolif icacy during the seasonal anoestrous (Haresign et al., 1990;Haresign, 1992;Abecia et al., 2007).Those effects are associated with an improvement in ovulation rate (Zúñiga et al., 2002), luteal function (Durotoye et al., 1997;Abecia et al., 2002) and embryo viability (Forcada et al., 2006).For a summary of the effects of exogenous melatonin on the ovary and early embryos in ewes, see Abecia et al. (2008).
The high amphilicity of melatonin promotes its rapid transfer into organs and fluids, and this pineal hormone can rapidly pass through cellular membranes.Reported increases in prolificacy subsequent to the administration of melatonin in sheep might be due to the direct effect of exogenous melatonin on the ovine oocyte, which has been observed in other species (Ishizuka et al., 2000;Na et al., 2005;Parka et al., 2006).Several studies have reported the presence of melatonin in human pre-ovulatory follicular fluid and the seasonal variation of its contents (Brzezinski et al., 1987;Yie et al., 1995), and melatonin receptors have been found on rat, mouse, and porcine ovarian cells (Soares et al., 2003;Na et al., 2005;Kang et al., 2008), which suggests that melatonin has a direct effect on oocyte competence.
In sheep, some studies have suggested that seasonal anoestrous can have a significant detrimental effect on the number of recovered oocytes per female, the competence of the oocytes, and the in vitro fertilization rate (Stenbak et al., 2001;Vázquez et al., 2009); however, information about the effects of melatonin treatments on oocyte quality and in vitro fertilization (IVF) during anoestrous is limited.In one study, after IVF of the oocytes recovered from superovulated ewes in the nonreproductive season, melatonin-treated and untreated ewes did not differ significantly in their fertilization rates (Luther et al., 2005).In another study, developmental competence of oocytes collected from ewes during the anoestrous was significantly improved by supplemental exogenous melatonin (Valasi et al., 2006).Although there are reports of the positive effects of adding melatonin in in vitro maturation (IVM) (Dimitriadis et al., 2005) and embryo culture media (Ishizuka et al., 2000;Na et al., 2005), the effect of melatonin in the IVM and IVF media on the develop-mental competence of ovine oocytes has not been evaluated thoroughly, which is needed to improve the efficiency of IVM, IVF, and in vitro embryo culture procedures.
The objective of this study was to evaluate the effects of two different melatonin concentrations on the in vitro maturation and fertilization of oocytes from sheep ovaries collected at the slaughterhouse, and on the in vitro culture of the embryos.

Oocyte collection and in vitro maturation
From November to February, the ovaries of mature Rasa Aragonesa ewes were collected at the slaughterhouse and transported to the laboratory in phosphatebuffered saline solution (PBS) supplemented with 100 IU mL -1 of penicillin-G and 100 µg mL -1 of streptomycin sulphate at 35-37°C.Except where indicated otherwise, all of the reagents for the preparation of the media were purchased from Sigma-Aldrich Co.
Oocytes were collected using a combination of puncture and slicing techniques (Wani et al., 1999) in a Petri dish that contained handling medium (Hepesbuffered TCM-199 supplemented with 0.1% of polyvinyl alcohol, 0.04% of sodium bicarbonate, 25 IU mL -1 of heparin, 100 IU mL -1 of penicillin-G, and 100 µg mL -1 of streptomycin sulphate).Following Wani et al. (2000), oocytes were categorized based on their cumulus cells and cytoplasm morphology; only the oocytes that had several layers of cumulus cells and a uniform cytoplasm were selected for in vitro maturation.Those oocytes were randomly assigned either to one of two melatonin-treatment groups [10 -5 M (M5) or 10 -6 M (M6)] or to one of two untreated (control) groups [C5 (control group for embryos treated with 10 -5 M melatonin) and C6 (treated with 10 -6 M melatonin)].and transferred into a maturation medium that contained bicarbonate-buffered TCM-199, supplemented with 10% (v/v) oestrus sheep serum, 10 µg mL -1 each of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), 100 µM of cysteamine, 0.3 mM of sodium piruvate, 100 IU mL -1 of penicillin G, and 100 µg mL -1 of streptomycin sulphate, covered with mineral oil, and incubated for 24 h at 39°C under 5% CO 2 and a saturated humidity.
Melatonin was dissolved in dimethyl sulfoxide (DMSO) and PBS to produce concentrations of 10 -5 or 10 -6 M, which was administered to the oocytes in the M5 and M6 groups, respectively.The final concentration of DMSO in the media was 0.01% v/v, with no effect on embryo development, as stated by Ishizuka et al. (2000).

In vitro fertilization and embryo culture
After the end of incubation, oocytes were freed from the cumulus cells and transferred to a fertilization medium that consisted of synthetic oviductal fluid (SOF) without glucose (Tervit and Whittingham, 1972), supplemented with 2% (v/v) of oestrous sheep serum, 10 µg mL -1 of heparin, and 1 µg mL -1 of hypotaurine.Again, melatonin was added to the fertilization medium of the M5 and M6 groups in the concentrations used in the maturation medium.
On the day of fertilization, fresh semen was collected from four Rasa Aragonesa rams, pooled, diluted 1:10 in a saline medium that contained 0.25 M sucrose, 10 mM Hepes, 2 mM KOH, 5 mM glucose, 0.5 M sodium phosphate monobasic, and 100 mM ethylene glycol tetraacetic acid (EGTA), and kept at 15°C until needed for fertilization.Highly motile spermatozoa were selected using the swim-up technique, added to the fertilization medium (350 µL) containing the oocytes at a final concentration of 1 × 10 6 spermatozoa mL -1 , covered with mineral oil, and incubated for 24 h, at 39°C in 5% CO 2 .
After 24 and 36 h, the cleaved embryos in each group were randomly divided into two sets.Half of the embryos in each group were cultured in a media that contained melatonin at either a 10 -5 M concentration (embryos from the M5 and C5 oocytes) or a 10 -6 M concentration (embryos from the M6 and C6 oocytes), and the other half remained as untreated controls.The embryos from the eight groups (M5M, M5C, C5M, C5C, M6M, M6C, C6M, and C6C) were placed in a culture medium that contained SOF supplemented with essential and nonessential amino acids at oviductal concentrations (Walker et al., 1996), 0.4% BSA (w/v), 1 mM of L-glutamine, 100 IU mL -1 of penicillin G, and 100 µg mL -1 of streptomycin sulphate, covered with mineral oil, and kept at 39ºC in a maximally humidified atmosphere, with 5% CO 2 , 5% O 2 , and 90% N 2 until the blastocyst stage (day 8).The number and developmental stage of blastocysts (young, expanded, hatching and hatched blastocysts) were recorded at 6, 7, and 8 days after fertilization.
Non-cleaved oocytes were observed under stereomicroscope to assess their maturation stage.Ooocytes showing the first polar body were considered matured, and oocytes with two polar bodies were considered fertilized, but not cleaved.

Statistical analysis
Maturation and cleavage rates were calculated over the number of selected oocytes, whereas fertilization rate was calculated over the number of matured oocytes, and blastocysts rates were calculated over the number of cleaved embryos.
Maturation, cleavage, and blastocyst rates of the experimental groups were compared by means of chisquare test (SPSS Software, v.14.0).When at least one value had an expected frecuency less than 5, Yates' correction for continuity was used.

Results and discussion
In this study, the lower of the two melatonin concentrations (10 -6 M) increased significantly the maturation rate of Rasa Aragonesa oocytes (P < 0.05) and tended to increase the cleavage rate (P < 0.1); however, a 10 -5 M concentration of melatonin in the media did not influence significantly the maturation, fertilization or cleavage (Table 1).The addition of melatonin to IVM and IVF media can improve in vitro oocyte maturation and cleavage rates in bovids (Dimitriadis et al., 2005), mice (Na et al., 2005) and humans (Parka et al., 2006).
High concentrations of melatonin in follicular fluid (Brzezinski et al., 1987), and the presence of receptors in granulose cells (Soares et al., 2003;Na et al., 2005;Kang et al., 2008), suggest that melatonin might be important to ovarian function.At night, the melatonin concentrations in physiological plasma in Rasa Aragonesa ewes are about 4 × 10 -7 M (Forcada et al., 1995).Studies have shown that physiological melatonin concentrations in human follicular fluid are almost three times higher than they are in the serum (Brzezinski et al., 1987;Ronnberg et al., 1990).Thus, the melatonin concentration of 10 -6 M could provide a basis for evaluating the benefits of exogenous melatonin at the gamete and early embryo stages.Furthermore, the beneficial effect of melatonin on early embryo development might be mediated through the reactive oxygen species (ROS) scavenger properties of the pineal hormone (Reiter et al., 2000).As a ROS scavenger, melatonin might be very valuable shortly after IVF because high concentrations of spermatozoa in small volumes of IVF medium lead to an increase in free radicals.Oxidative stress has toxic effects and melatonin provides protection against the oxidative stress (Tamura et al., 2008).
These results and those of others (Adriaens et al., 2006) suggest that the effect of melatonin on oocyte maturation is dose-dependent.Although melatonin toxicity is reported to be extremely low, oocyte maturation in female mice was significantly impaired by melatonin concentrations of 10 -3 M or higher.In the early stages of maturation, melatonin has an even greater effect on the in vitro maturation of oocytes (Dimitriadis et al., 2005).
In this study, blastocyst rates did not differ significantly among groups at either of the two melatonin concentrations used (Table 1) but the highest dose added to the culture medium of the control group seemed to impair embryo development.When data were analyzed by culture day and blastocyst type, significant differences were found on hatching blastocyst rate in day 8 between 10 -5 M treated and control groups, and on young blastocyst rate in day 8 derived from oocytes matured with or without 10 -6 M melatonin and cultured without the pineal hormone (Table 2).Elsewhere, another study demonstrated a negative effect of melatonin on blastocyst rate when it was added to the maturation medium at 10 -5 M concentration (Casao et al., 2007), but a concentration halfway between 10 -6 and 10 -5 M (0.43 • 10 -5 M) in the culture medium appeared to increase the hatching rate of thawed ovine blastocysts (Abecia et al., 2002).
In general, the effects of melatonin on in vitro preimplantational embryo development are unclear.Several studies have shown that enriching the culture medium with melatonin can improve embryo development, but blastocyst rates can vary widely depending on the melatonin concentrations (Ishizuka et al., 2000;Danilova et al., 2004;Manjunatha et al., 2008).When embryos are subjected to melatonin concentrations of 10 -3 M or higher, cleavage rates can be impaired (Rodriguez-Osorio et al., 2007), but other studies covering a wide range of concentrations suggest that melatonin treatments do not influence the preimplantation development of embryos (McElhinny et al., 1996;Tsantarliotou et al., 2007).
In addition to its benefits to in vitro maturation and early embryo development, melatonin might have a benef icial effect on embryo preimplantation development because of its capacity as a radical scavenger (Chetsawang et al., 2006), to protect embryonic cells from oxidative stress.High levels of reactive oxygen species can damage spermatozoa, oocytes, and embryos (Agarwal et al., 2003), and in vitro melatonin treatments might protect them against oxidative stress.In a recent study, Papis et al. (2007) showed that the beneficial effects of melatonin on bovine embryo development were greater when embryos were cultured in the presence of high concentrations of atmospheric oxygen (20%) rather than at physiological concentrations (7%).In addition, melatonin can increase blastocyst rates and blastocyst total cell number concomitant to a significant decrease of apoptotic nuclei rate in preimplantational parthenogenetic porcine embryos (Choi et al., 2008).In conclusion, this study demonstrated that adding 10 -6 M melatonin to IVM and IVF media increased in vitro maturation and early cleavage rates of sheep oocytes.Although a higher concentration (10 -5 M) of melatonin did not have the same effect, additional studies are needed to optimize the use of melatonin in IVM, IVF, and in vitro embryo culture procedures.

Table 1 .
Maturation, fertilization, cleavage and blastocyst rate of oocytes from Rasa Aragonesa ewes matured and/or cultured with or without 10 -5 or 10 -6 mol L -1 melatonin Within columns, different letters (a, b) indicate differences of P < 0.05 at the same treatment dose.Within columns, different letters (c, d) indicate differences of P = 0.08 at the same treatment dose.

Table 2 .
Blastocyst type rate of oocytes from Rasa Aragonesa ewes matured and/or cultured with or without 10 -5 or 10 -6 mol L -1 melatonin, on days 6, 7 and 8 after oocyte fertilization Within columns, different letters (a, b) indicate differences of P < 0.05 at the same treatment group.