ORIGINAL ARTICLE

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos 4

n1, M. E. Arias1, J. Risopatro
n1,2, R. Felmer1,2,3, J. Alvarez
nchez1,6 C. Cheuquema , T. Mogas5 & R. Sa


n (BIOREN-CEBIOR), Facultad de Medicina, Universidad de La Frontera, Temuco, Chile; 1 Centro de Biotecnolog
ıa de la Reproduccio 2 Departamento de Ciencias B
asicas, Universidad de La Frontera, Temuco, Chile;
micas y Recursos Naturales, Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, 3 Departamento de Ciencias Agrono Temuco, Chile; ~a, Spain; 4 Centro ANDROGEN, La Corun noma de Barcelona, Bellaterra, Spain; 5 Departamento de Medicina i Cirurgia Animals, Universitat Auto 6 Departamento de Ciencias Precl
ınicas, Facultad de Medicina, Universidad de La Frontera, Temuco, Chile

Keywords Gamete co-incubation—in vitro fertilisation— melatonin—oxidative stress—sperm function Correspondence
l S
Dr Rau anchez, Facultad de Medicina, Universidad de La Frontera, Calle Montevideo # 0870, Temuco, Chile. Tel.: +56 45 2744248; Fax: +56 45 2234326; E-mail: [email protected] Accepted: May 16, 2014 doi: 10.1111/and.12308

Summary Gamete co-incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co-incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2
6.7% versus 1 mmol: 2.0
1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.

Introduction In vitro embryo production techniques are a challenge for scientist trying to determine basic requirements for successful embryo development (Besenfelder et al., 2010). The quality of in vitro produced embryos is lower than those obtained in vivo, and the morphological, physiological and biochemical differences between in vitro and in vivo embryo production are described extensively in literature (Rizos et al., 2002; Lonergan & Fair, 2008). Gametes are highly susceptible to reactive oxygen species (ROS) attack during in vitro manipulation in assisted reproductive techniques (ART) because they are often © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–12

exposed to supra-physiological levels of ROS (Du Plessis et al., 2008). During in vitro fertilisation (IVF), co-incubation of spermatozoa and oocytes generates high levels of free radicals, due to surrounding dead spermatozoa close to growing zygotes. This may affect viability and subsequent embryo quality (Nedambale et al., 2006; Silva et al., 2007). Previous research has evaluated the addition of different antioxidants to counteract the deleterious effect of oxidative stress on IVF in cattle. However, added concentrations of different antioxidants do not show any beneficial effects (Ali et al., 2003; Goncalves et al., 2010; Marques et al., 2010). In contrast, melatonin exhibits the 1

Melatonin in bovine IVF and sperm function

most desirable characteristics of a good antioxidant, scavenging a wide variety of free radicals (Galano et al., 2011). Its protective action has been described for different reproductive biotechnologies such as in vitro oocyte maturation in buffalo (Manjunatha et al., 2009) and cattle (Tsantarliotou et al., 2010; Takada et al., 2010, 2012; Sampaio et al., 2012) improving sperm functionality in buffalo (Li et al., 2012), ram (Kaya et al., 2000; Casao et al., 2010), boar (Jang et al., 2010), mouse (Sarabia et al., 2009), hamster (Fujinoki, 2008) and human spermatozoa (Espino et al., 2010), stimulating embryo development in mice (Ishizuka et al., 2000; Gao et al., 2012), buffalo (Manjunatha et al., 2009) and cattle (Papis et al., 2007) and improving sheep embryo survival (Abecia et al., 2002). However, limited research has examined its impact on bull sperm functionality, especially during the incubation time of IVF protocol. Research has also not been conducted to assess the impacts of its application during gamete co-incubation and its subsequent effect on in vitro embryo development in cattle. The present research was conducted to determine (i) the impact of melatonin on reducing free radicals during bovine IVF and subsequent embryo development and (ii) the effect of melatonin on bull sperm function using the same incubation time in which spermatozoa are exposed during the IVF protocol. Materials and methods Ethics statement This study was approved by the Institutional Review Board of the Faculty of Medicine at the Universidad de La Frontera, Temuco, Chile. All animal and in vitro procedures were conducted according to Chilean law No. 20.380 on Animal Protection (http://www.leychile.cl/ Navegar?idNorma=1006858). Frozen bull semen was obtained from the Artificial Insemination Center, Austral University, Valdivia, Chile (http://www.uach.cl/centro/inseminacionartificial/). Bovine ovaries were obtained from a slaughter house (Frigor
ıfico Temuco, Temuco, Chile; http://www.aasa.cl/empresasaasa/comercial/frigorifico-temuco-s-a/). In vitro embryo production Experimental design Melatonin supplementation was made during gamete coincubation. It was diluted in absolute ethanol (8 mg ml1) (Abecia et al., 2002) and kept at 20 °C until use. For each experiment, successive dilutions of melatonin in IVF-TALP were prepared. In Experiment 1, 0.01, 0.1 and 1 mmol melatonin and in Experiment 2, 10, 2

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100 and 1000 nmol melatonin were introduced. In all experiments, a culture well without melatonin was used as a control. On preliminary analysis, a culture well with the highest ethanol concentration was made as a vehicle control, but we did not observe any effect, so in subsequent experiments, this was not included (data not show). After IVF, presumptive zygotes from each treatment were cultured separately. Oocyte collection and in vitro maturation (IVM) Ovaries were collected from a local slaughterhouse (Frigor
ıfico Temuco, Chile). Cumulus–oocyte complexes (COC) were aspirated from 2- to 7-mm follicles using an 18-gauge needle attached to a 10-ml syringe. Good-quality oocytes having a corona of cells of at least four layers and a uniformly granulated cytoplasm were selected and matured in 400 ll TCM-199 medium (50 COC per well), supplemented with 10% inactivated FBS (Hyclone Laboratories, Inc., South Logan, UT, USA) and 6 mg ml1 LH hormone (Sioux Biochemical, Inc., Sioux City, IA, USA), 6 mg ml1 FSH hormone (Sioux Biochemical), 1 mg ml1 estradiol and 0.2 mmol pyruvate and then incubated for 22–24 h at 38.5 °C in 5% CO2 and a humidified atmosphere. A total of 2600 oocytes were cultured in this research: 600 oocytes were distributed in four treatments (n = 3) at Experiment 1, and for Experiment 2, we cultured 2000 oocytes distributed in four treatments (n = 10). Semen preparation Frozen bovine semen (20–25 9 106 spermatozoa/0.5-ml straw) obtained from Artificial Insemination Center UACH (Austral University, Valdivia, Chile) was used. Straws were thawed in a water bath (38 °C for 30 s). Spermatozoa were selected through a Percoll gradient (90–45%) and washed by centrifugation at 300 g with sp-Talp medium. In vitro fertilisation Matured oocytes and spermatozoa were co-incubated for 18–20 h in a four-well culture plate containing 400 ll IVF-TALP supplemented with 0.2 mmol sodium pyruvate, 6 mg ml1 fatty acid-free BSA and 10 lg ml1 gentamicin sulphate (Parrish et al., 1986). Final IVF-TALP contained PHE (penicillamine 0.08 mmol, hypotaurine 0.04 mmol and epinephrine 0.01 mmol), 2 lg ml1 heparin and 106 ml1 Percoll-separated frozen–thawed spermatozoa. At this step, we introduced melatonin treatments (Exp. 1 and Exp. 2). Embryo culture Presumptive zygotes were stripped of cumulus cells by vortex and cultured in 50 ll drops (25 embryos per drop) of KSOM (Embryo Max; Millipore Corp, Billerica, © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–12

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MA, USA) with 0.4% FAF-BSA for 3 days and then KSOM 10% FBS until day 7 under mineral oil at 38.5 °C and 5% CO2, 5% O2 and 90% N2 in a humidified atmosphere. Embryo quality Expanded blastocysts from 7-day culture of each treatment from Experiment 2 were incubated with 10 lg ml1 of Hoechst for 10 min and then mounted in 10 ll of PBS/glycerol. Total cell number was analysed under an epifluorescent microscope (Axiolab-ZEIZZ, Jena, Germany). IMAGE J software (NIH, Bethesda, MD, USA) was used for embryo cell counting. Sperm evaluation Experimental design In Experiment 3, thawed semen from the same bull was selected through a Percoll gradient for IVF procedures and was aliquoted at a final concentration of 106 cells ml1 in IVF-TALP. Melatonin was diluted as in Experiment 2. In preliminary assays, a vehicle control was made with the highest ethanol concentration used, but we did not observe any effects, so in the subsequent experiments, it was not included (data not show). Aliquots of control and treatments (10, 100 and 1000 nmol melatonin) were immediately evaluated at zero time (T0), and remaining aliquots were incubated at 38.5 °C and 5% CO2 in a humidified atmosphere for 4 (T4) and 18 h (T18), and then, all functional sperm parameters were evaluated. Motility Analysis was performed using an integrated sperm analysis system V1.0 (Proiser, Valencia, Spain). Samples were maintained at 37 °C on a heated platen. DC20 chambers were filled with 2.7 ll of sperm solution. This system analysed 25 consecutive and digitised photographic images obtained from a single field at 109 magnification and negative phase-contrast field. Five separate fields were taken from each sample for total (MOT, %) and progressive (PROG, %) motilities; average path (VAP, lm s1), straight line (VSL, lm s1) and curvilinear (VCL, lm s1) velocities; amplitude of lateral head displacement (ALH, lm); beat/ cross-frequency (BCF, Hz); straightness (STR, %); linearity (LIN, %) and wobbler coefficient (WOB, %). Viability and plasma membrane integrity This was detected using SYBR-14/PI (LIVE/DEADâ Sperm Viability kit; Molecular Probes L-7011, Eugene, OR, USA) according to the manufacturer’s instructions. For each tube, a volume of 2 ll of SYBR-14 (100 nm l1 final concentration) and 2 ll of propidium iodide (PI)

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Melatonin in bovine IVF and sperm function

(stock solution 2.4 mmol) were added to a final volume of 400 ll sperm suspension in IVF-TALP (106 cells ml1) and incubated for 8 min at 37 °C in the dark and immediately analysed by flow cytometry. Spermatozoa were classified as viable with an intact plasma membrane (PI/SYBR-14+), dead (PI+/SYBR-14) or moribund (PI+/SYBR-14+). Acrosome membrane integrity This was assessed with FITC-conjugated Pisum Sativum Agglutinin (FITC-PSA)/PI according to the manufacturer’s instructions (kit FITC-PSA/PI; SIGMA, St Louis, MO, USA). For each tube, 20 ll of FITC-PSA (from 1 mg ml1 stock) and 2 ll of PI (stock solution 2.4 mmol) were added to a final volume of 400 ll of sperm suspension in IVF-TALP (106 cells ml1) followed by 8-min incubation at 37 °C in the dark and immediately analysed by flow cytometry. Spermatozoa were classified as viable with intact acrosome membrane (FITC-PSA/PI) or viable with damaged acrosome membrane (FITC-PSA+/PI), dead with intact acrosome membrane (FITC-PSA/PI+) or dead with damaged acrosome membrane (FITC-PSA+/PI+). Mitochondrial membrane potential (w) This was assessed with Rhodamine 123 (R-302; Molecular Probes, Eugene, OR, USA) diluted in dimethyl sulfoxide (DMSO). For each tube, 4 ll of Rhodamine 123 and 2 ll of PI (stock solution 2.4 mmol) were added to a final volume of 400 ll of sperm suspension in IVF-TALP (106 cells ml1) followed by incubation for 30 min at 37 °C in the dark and immediately analysed by flow cytometry. Spermatozoa were classified as viable with high mitochondrial membrane potential (ROD 123+/PI) or viable with low mitochondrial membrane potential (ROD 123/ PI), dead with high mitochondrial membrane potential (ROD 123+/PI+) or dead with low mitochondrial membrane potential (ROD 123/PI+). Reactive oxygen species We used Carboxy-H2DFFDA (C13293, Invitrogen, Eugene, OR, USA) fluorescent dye to estimate the intracellular ROS level in spermatozoa. For each tube, 2 ll of CarboxyH2DFFDA (0.01 mmol final concentration) and 2 ll of PI (stock solution 2.4 mmol) were added to a final volume of 400 ll of sperm suspension in IVF-TALP (106 cells ml1) followed by incubation for 30 min at 37 °C in the dark and immediately analysed by flow cytometry. Data were expressed as the percentage of dead (CH2DFFDA+/PI+) and viable (CH2DFFDA+/PI) spermatozoa with intracellular ROS and by the geometric mean (Gmean) of the fluorescence intensity (FI) in total spermatozoa.

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Melatonin in bovine IVF and sperm function

DNA fragmentation This was evaluated by terminal deoxynucleotidyl transferase UTP nick-end labelling (TUNEL), using a TUNEL assay according to manufacturer’s instructions (In Situ Cell Death Detection Kit with Fluorescein; Rocheâ, Mannheim, BW, Germany). Sperm suspension in PBS (106 cell ml1) was fixed in 4% formaldehyde for 15 min at 4 °C. They were then washed twice in PBS and permeabilised in 0.2% Triton X-100 and 0.1% of citrate solution for 30 min at room temperature. Samples treated with (DNase I, RNase-free, Fermentas, Vilnius, Lithuania, EU) for 10 min at 37 °C were made as positive controls. All samples were incubated with 5 ll of enzyme solution and 45 ll of the label solution for 60 min in a humid chamber at 37 °C in the dark. Negative controls were incubated in only 50 ll of label solution. After washing, a positive control for PI and all experimental samples were stained with 2 ll of PI (2.4 mmol stock solution) and resuspended in 400 ll of PBS and immediately analysed by flow cytometry. Data were expressed as percentage of spermatozoa with fragmented DNA (TUNEL+) or intact DNA (TUNEL). DNA oxidation We detected 8-hydroxy-20 -deoxyguanosine formation using the OxyDNA test (Argutus Medical No: BIO81DNA). Sperm suspensions in PBS (1 9 106 cell ml1) were fixed in 4% formaldehyde for 15 min at 4 °C. Positive controls were previously incubated in 4 M H2O2 for 1 h at 37 °C. Then, all samples were washed twice in PBS and permeabilised in 0.2% Triton X-100 and 0.1% citrate solution for 15 min at room temperature. Samples were washed in wash solution, then washed in PBS and incubated with 50 ll of binding protein-FITC conjugate, followed by 1-h incubation at room temperature in the dark. Then, they were washed and re-suspended in 400 ll of PBS and immediately analysed by flow cytometry. Data were expressed as the percentage of spermatozoa with 8hydroxy-20 -deoxyguanosine (OxyDNA+) and intact DNA (OxyDNA). Flow cytometry analysis This was performed in a BD FACS Canto IITM Flow Cytometer (BD Biosciences, San Jos
e, California, USA) (April 2010) SN: V96101286, (USA) using BD FACS DIVATM software (updated for version 6.0). For each sample, 10 000 events were captured. Statistical analysis Differences between experimental groups were measured using a one-way ANOVA test for cleavage, embryo development and total embryo cell count, and two-way ANOVA and Fisher’s test (LSD) for sperm functionality. P < 0.05 4

was considered statistically significant. All analyses were performed with STATGRAPHICS plus 5.1 version software (Warrenton, USA). Results Effects of melatonin on IVF and subsequent embryo development In Experiment 1, gametes were exposed to 0.01, 0.1 and 1 mmol of melatonin. No significant differences in cleavage rate were found compared to controls (C: 78.0
4.7%; 0.01 mmol: 70.2
7.8%; 0.1 mmol: 69.0
8.7% and 1 mmol: 50.1
27.3%) (P > 0.05). However, a significant decrease in blastocyst rate was observed at 1 mmol melatonin (C: 23.2
6.7% versus 1 mmol: 2.0
1.7%) (P < 0.05) (Table 1). In Experiment 2, in which gametes were exposed to 10, 100 and 1000 nmol melatonin, no differences on either cleavage rates (C:75.3
10.1%; 10 nmol: 74.7
12.1%; 100 nmol: 69.8
9.3%; 1000 nmol: 77.1
8.3%) or blastocyst rates were found (C: 22.9
6.3%; 10 nmol: 20.8
9.8%; 100 nmol: 18.6
10.2%; 1000 nmol: 20
10%) (P > 0.05) (Table 2). Total embryo cell count was assessed in 7-day expanded blastocyst from Experiment 2, but no statistical differences were found between melatonin exposure and controls (C: 151.3
52.1; 10 nmol: 145.9
49.7; 100 nmol: 149.3
48.1; 1000 nmol: 162.5
51.5) (P > 0.05) (Table 3 and Fig. 1). Effects of melatonin on sperm functionality In Experiment 3, spermatozoa were exposed to 10, 100 and 1000 nmol melatonin and evaluated at 0, 4 and 18 h after incubation (n = 4). Total (TOT, %) and progressive (PROG, %) motilities, average path (VAP, lm s1), straight line (VSL, lm s1), curvilinear (VCL, lm s1) velocities, amplitude of lateral head displacement (ALH, lm), beat/cross-frequency (BCF, Hz), straightness (STR, %) and linearity (LIN, %) decreased through incubation times (0, 4 and 18 h) and did not show any statistically significant differences between treatments (P < 0.05) (Table 4). Melatonin at 10 nmol showed a significantly greater wobbler coefficient (WOB, %) than samples with 1000 nmol melatonin (LSD media: 10 nmol: 66.0% versus 1000 nmol: 61.1%) (P < 0.05) (Fig. 2), but this parameter exhibited no differences across incubation times (P > 0.05). Viability and plasma membrane integrity (T0: 87.5
0.2%, T4: 39.6
8.1%, T18: 20.4
2.1%) (Fig. 3) and viable spermatozoa with intact mitochondrial membrane potential (w) decreased through incubation time (T0: 74.7
1.7%, T4: 51.5
1.8%, T18: 25.7
2.9%) © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–12

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Table 1 Cleavage and blastocyst development rates (%) with high melatonin concentration Treatment

No. zygotes

Cleavaged, % (mean
SD)

Control 0.01 mmol melatonin 0.1 mmol melatonin 1 mmol melatonin

91 86 89 89

71 60 62 44

(78.0 (70.2 (69.0 (50.1







4.7) 7.8) 8.7) 27.3)

Blastocysts, % (mean
SD) 21 (23.2 5 (5.7 9 (9.6 1 (2.0







6.7)a 4.9)a 6.0)a 1.7)b

Values with different superscript letters within the same column differ (P < 0.05). Table 2 Cleavage and blastocyst development rates (%) with lower melatonin concentration

Treatment Control 10 nmol melatonin 100 nmol melatonin 1000 nmol melatonin

No. zygotes

No. cleavaged, % (mean
SD)

No. blastocysts, % (mean
SD)

493 460

372 (75.3
10.1) 343 (74.7
12.1)

112 (22.9
6.3) 98 (20.8
9.8)

478

335 (69.8
9.3)

92 (18.6
10.2)

470

358 (77.1
8.3)

92 (20
10)

Table 3 Total cell count in day 7 expanded blastocysts

Treatment

No. examined embryos

Total cell count (mean
SD)

Control 10 nmol melatonin 100 nmol melatonin 1000 nmol melatonin

9 7 8 11

151.3 145.9 149.3 162.5

(a)

(b)

(c)

(d)







52.1 49.7 48.1 51.5

(Fig. 4), but there was no statistical difference between melatonin treatments in both functional parameters (P > 0.05). Viable spermatozoa with intact acrosome membranes were negatively influenced by incubation time (T0: 53.6
5.1%, T4: 27.4
1.4%, T18: 19.3
2.9%; P < 0.05) (Fig. 5a); 1000 nmol melatonin showed a significantly lower percentage of viable spermatozoa with intact acrosomes than 10 nmol melatonin and controls (LSD media; 1000 nmol: 30% versus 10 nmol: 33.8% and C: 33.4%) (P < 0.05) (Fig. 5b). Viable spermatozoa with intracellular ROS increased significantly through incubation time (T0: 0%, T4: 0%, T18: 0.1%) (P < 0.05) (Fig. 6a). Treatment with 1000 nmol melatonin showed a lower percentage of viable spermatozoa with intracellular ROS versus 10 nmol melatonin (LSD media; 1000 nmol: 0.025% versus 10 nmol: 0.05%) (P < 0.05) (Fig. 6b). Likewise, Gmean of fluorescence intensity from total spermatozoa increased through incubation time (T0: 0, T4: 62.8, T18: 161) (Fig. 7a), and 10 nmol melatonin samples were significantly greater than the control (P < 0.05) (LSD media; 10 nmol: 114.1 versus C: 74.6 respectively) (Fig. 7b). The DNA fragmentation index remained unchanged through incubation time (T0: 0.4
0.1%, T4: 0.34
0.2%, T18: 0.5
0.4%), and 1000 nmol melatonin samples showed significantly more DNA fragmentation than controls (P < 0.05) (LSD media; 1000 nmol: 0.85% versus C: 0.42% respectively) (Fig. 8). The DNA oxidation percentage remained constant through the incubation time (T0: 29.5
9%, T4: 31.7
15%, T18: 28.7
16%), and 1000 nmol melatonin samples showed significantly more DNA oxidation than controls (LSD media; 1000 nmol: 42.2% versus C: 29.9% respectively) (P < 0.05) (Fig. 9). Discussion

Fig. 1 Representative images of 7-day expanded blastocyst from Experiment 2. Total embryo cells with Hoechst 33342 staining. (a) Control, (b) 0.01 mmol melatonin, (c) 0.1 mmol melatonin and (d) 1 mmol melatonin.

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Oxidative stress control has a physiological impact on reproductive processes; low free radical levels modulate gamete function, particularly capacitation and chemotactic acquisition by spermatozoa (Sanchez et al., 2010), sperm-mediated oocyte activation, embryonic genome activation and blastocysts hatching from the zona 5

6

Control 10 nmol melatonin 100 nmol melatonin 1000 nmol melatonin Control 10 nmol melatonin 100 nmol melatonin 1000 nmol melatonin Control 10 nmol melatonin 100 nmol melatonin 1000 nmol melatonin

0

Values are mean
SD.

18

4

Treatment

Time (h) 83.6 87.2 76.6 77.6 35.9 30. 8 28.8 33.1 16.9 14.1 15.9 17.2













8.4 0.1 5.5 7.4 8.1 5.3 7.7 13.3 6.1 4.5 4.1 1.5

MOT (%) 52.1 56.1 41.6 46.5 18.8 16.9 17.8 23.6 8.9 8.7 7.0 6.7













11.7 11.6 6.6 10.3 7.9 5.9 7.8 10.1 2.5 4.8 4.2 0.9

PROG (%) 111.4 113.7 103.0 102.5 75.8 76.7 77.7 88.3 69.3 60.1 66.9 62.4













12.3 9.3 9.5 6.1 26.8 9.9 10.5 12.1 10.1 14.4 11.2 7.8

VCL (lm/s) 59.7 58.7 50.3 50.3 37.7 36.6 38.9 45.0 30.8 31.4 30.6 24.5













13.3 11.3 9.3 7.2 14.5 7.0 9.3 8.0 7.0 8.7 8.3 4.8

VSL (lm/s) 75.8 74.4 65.3 65.7 48.3 48.5 48.7 54.9 41.2 41.1 42.4 35.8













12.8 9.3 8.3 6.0 16.5 4.7 9.6 9.7 8.9 9.1 7.6 4.6

VAP (lm/s) 78.1 78.5 76.5 76.4 77.7 75.2 79.5 82.1 74.7 75.9 71.3 68.1















5.9 6.4 4.5 4.7 6.8 12.2 8.9 4.5 6.6 4.2 4.9 4.3















51.33 51.4 48.6 49.0 50.2 48.6 50.1 50.9 44.3 52.0 44.9 39.1

STR (%)

LIN (%)

Table 4 Motility rate and kinematic parameters of bull spermatozoa at 0, 4 and 18 h of incubation with different melatonin concentrations

4.0 5.8 4.4 4.1 6.4 9.9 6.1 4.2 4.7 6.1 7.6 5.7

65.4 65.2 63.3 64.1 64.4 63.9 62.6 61.9 59.4 68.9 63.1 57.4













4.1 3.4 2.5 2.8 4.5 8.0 6.9 3.5 8.4 8.1 3.1 1.9

WOB (%) 3.9 3.9 3.8 3.8 2.4 2.7 2.5 3.2 2.0 2.0 1.9 2.4















0.1 0.1 1.3 0.2 1.2 0.4 0.4 0.8 0.8 0.9 1.0 0.8

ALH (lm)

12.4 12.5 11.4 11.7 8.9 9.7 9.1 10.5 7.5 7.5 8.3 8.5















1.5 1.4 1.6 0.9 4.8 2.5 1.7 2.7 3.7 2.9 3.4 2.2

BCF (Hz)

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Fig. 2 Effect of melatonin on wobbler coefficient (WOB %). Fisher’s least significant difference (LSD).

Fig. 3 Effect of melatonin on viability and plasma membrane integrity through incubation time.

Fig. 4 Effect of melatonin on mitochondrial membrane potential (w) through incubation time.

pellucida (Harvey et al., 2002), which are basic elements of fertilisation process. A supra-physiological level of reactive oxygen species (ROS) has been associated with deleterious effects due to destruction or alteration of proteins, lipids and nucleic acids (Ma, 2010). It has been reported that hydrogen peroxide and superoxide anion overproduction cause direct damage of mitochondria and subsequently activate proteases and caspases, resulting in chromatin condensation and fragmentation, apoptosis, altered cell division and embryonic developmental arrest (Velez-Pardo et al., 2007). Deleterious effects of ROS produced during gamete coincubation on IVF processes has been described, which subsequently has a negative effect on embryo quality (Nedambale et al., 2006; Enkhmaa et al., 2009). As a

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(a)

(b)

Fig. 5 Effect of melatonin on acrosome membrane. (a) Interaction with incubation time and (b) Fisher’s least significant difference (LSD).

Melatonin in bovine IVF and sperm function

(a)

(b)

Fig. 7 Effect of melatonin on fluorescence intensity (Gmean) from total spermatozoa with intracellular ROS. (a) Interaction with incubation time and (b) Fisher’s least significant difference (LSD).

(a)

(b) Fig. 8 Effect of melatonin on sperm DNA fragmentation %. Fisher’s least significant difference (LSD).

Fig. 6 Effect of melatonin on intracellular ROS. (a) Interaction with incubation time and (b) Fisher’s least significant difference (LSD).

result, it has been hypothesised that antioxidant supplementation of conventional fecundation media could avoid oxidative stress, thereby improving technique efficiency and embryo quality. Melatonin is a potent antioxidant molecule and shows protective capacities against molecular damage and cell death in gametes and embryos (Reiter et al., 2013) and is described as beneficial for human IVF and embryo transfer (Tamura et al., 2008). We test whether supplementing IVF media with melatonin improves bovine in vitro embryo production. This is the first investigation adding melatonin to bovine IVF and analysing its effect on sperm function. © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–12

Fig. 9 Effect of melatonin on sperm DNA oxidation %. Fisher’s least significant difference (LSD).

During Experiment 1, IVF media with 1 mmol melatonin significantly decreased blastocyst rates. These results are consistent with the literature, which shows that the same melatonin concentration delayed blastocyst rates in mice (Tian et al., 2010; Gao et al., 2012), decreased cleavage 7

Melatonin in bovine IVF and sperm function

and blastocyst rates in porcine embryo cultures (Rodriguez-Osorio et al., 2007), and was significantly deleterious when was added to porcine maturation media (Shi et al., 2009). Similar results have been reported with other antioxidants added to bovine IVF media in the same concentration range (Ali et al., 2003; Goncalves et al., 2010). It is described that physiological level of free radicals is needed for normal fertilisation (Harvey et al., 2002) and this melatonin concentration possibly generated a complete depletion of ROS that impairs embryo development. In other hand, an antiproliferative effect of melatonin is described in some cell tissues (Vijayalaxmi et al., 2002; Sanchez-Barcelo et al., 2003), including pro-apoptotic and pro-oxidant action that triggers NF-kB pathways (Cristofanon et al., 2009) and the stimulation of ROS production and reduced-GSH depletion at certain melatonin concentrations (Osseni et al., 2000). Accordingly, our results shown that 1 mmol melatonin has a deleterious effect during IVF because it subsequently damages embryo development. Lower concentrations of melatonin (Exp. 2: 10, 100, 1000 nmol and C) did not improve either in vitro embryo production or total cell count in 7-day expanded blastocyst. These results contrast with findings that demonstrated that increasing concentrations of melatonin from 0.01 to 100 nmol improve blastocyst and hatched blastocyst rates up to a maximum at 1 nmol melatonin in mice embryo cultures (Gao et al., 2012). Similarly, adding 10 and 1000 nmol melatonin to early pre-implantation mouse embryos enhanced development (Ishizuka et al., 2000) and concentrations from 0.01 to 1 nmol in porcine maturation media increased cleavage, blastocyst rates and total cell count of parthenogenetic blastocyst (Shi et al., 2009). Blastocyst formation is the first differentiation process during early embryonic development in mammals and is related with embryo quality (Koo et al., 2002). In this research, blastocyst rates and total cell number were similar between melatonin treatments and controls; hence, melatonin appears to have no effect on embryo quality. Apparently, melatonin supplementation of IVF at concentrations tested does not improve in vitro bovine embryo production. In Experiment 3, most sperm kinetic parameters evaluated, and viability and plasma membrane integrity, acrosome membrane integrity and mitochondrial potential decreased significantly through incubation time. Accordingly, results from Experiment 3 are consistent with the literature for bull spermatozoa function through co-incubation time during IVF (Goncalves et al., 2010) and other studies that evaluate sperm function (Pe~ na et al., 1999; Aziz & Enbergs, 2005; Bollwein et al., 2008; Awad, 2011). Sperm samples with 1000 nmol melatonin had a significantly lower wobbler coefficient (WOB, %), a lower 8

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percentage of viable sperm with intact acrosomes, a lower percentage of viable sperm with intracellular ROS, greater DNA fragmentation and higher DNA oxidation than controls, and total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls. A high wobbler coefficient (WOB, %) at 10 nmol melatonin may be related to sperm capacitation processes (Ramio et al., 2008) and effects of antioxidants such as have been described with ascorbic acid (Hu et al., 2010). Similarly, lower percentages of intact acrosomes that we observed at 1000 nmol melatonin are related with induction of capacitation processes and subsequent acrosome reaction. In this way, it has been described that melatonin at 0.1 nmol activated capacitation in ram spermatozoa (Casao et al., 2010) and 0.001 to 1000 nmol melatonin enhanced hyperactivation in hamster sperm (Fujinoki, 2008). In other hand, oxidative stress is associated with male infertility and specifically linked with DNA integrity alterations in spermatozoa (Zribi et al., 2011). In our results, spermatic intracellular ROS increase after co-incubation time that is required for IVF technique. We conclude that the increase of ROS in the IVF media culture is due to spermatozoa, as is described that ROS production increase during IVF and is not affected by oocyte interaction (Enkhmaa et al., 2009). Reduced intracellular ROS in sperm at 1000 nmol melatonin is associated with its widely described antioxidant action (Sariahmetoglu et al., 2003; Bustos-Obreg
on et al., 2010; Jang et al., 2010; Tsantarliotou et al., 2010; Espino et al., 2011). However, melatonin at 10 nmol seems to be a pro-oxidant molecule for total spermatozoa, like another substances described as antioxidants that at certain concentration generates an inverse effect, being prooxidants (Sakagami & Satoh, 1997; Hu et al., 2010). This can be explained by a dual effect of melatonin that is related both to antioxidant concentration used (Casao et al., 2010; Gomez et al., 2010) and cellular structure where its effects have been analysed (Letelier et al., 2010). Accordingly, melatonin can increase ROS levels and promote DNA oxidation at certain concentrations in bovine sperm, acting as pro-oxidant substance. Progressive oxidative damage processes begin with the excessive generation of ROS, leading to lipid peroxidation and oxidative DNA damage, and culminates in DNA fragmentation and death (Aitken et al., 2010). ROS might lead to base modifications by reaction with DNA nucleotides, particularly 8-hydroxy-20 -deoxyguanosine (8-oxoguanine) formation and DNA fragmentation as single- and double-strand DNA breaks (Aitken & Krausz, 2001). These two parameters are also positively correlated (De Iuliis et al., 2009; Zribi et al., 2011). Our results show that DNA damage (fragmentation and oxidation) © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–12

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remained similar through incubation time (18 h) and are according with kinetic DNA fragmentation described in bovine (Gonz
alez-Mar
ın et al., 2011) and boar spermatozoa, which only shows a significant increase in DNA fragmentation after 2-day incubation (Perez-Llano et al., 2010). Although it has been suggested that using antioxidants could prevent oxidative stress and subsequent sperm DNA damage (Aitken et al., 2010; Mukhopadhyay et al., 2011; Zribi et al., 2011), our results shown that DNA fragmentation and DNA oxidation at 1000 nmol melatonin were significantly higher than controls. At the same concentration, we observed less intracellular ROS in spermatozoa. Probably, intracellular ROS became to more toxic free radicals with a short half life time that prevented its detection by flow cytometry and then generated significantly more DNA damage at 1000 nmol melatonin. Hence, this concentration of melatonin is harmful for DNA integrity in bovine spermatozoa. Sperm DNA damage influences fertility (Loft et al., 2003; Kasimanickam et al., 2006) and a higher percentage of spermatozoa with 8-oxoguanine are associated with lower embryo quality after IVF or intracytoplasmic sperm injection (ICSI) (Meseguer et al., 2008). However, spermatozoa with significant DNA damage could retain the ability to fertilise if their membranes are still intact (Aitken et al., 1998). Accordingly, our results shown that despite sperm DNA damage at 1000 nmol melatonin, there were no differences in embryo production (cleavage and blastocyst rates, and total cell count) when we supplemented IVF media culture with that melatonin concentration. In this way, sperm DNA damage does not necessarily impair fertilisation because oocytes have DNA repair mechanisms that enable further embryo development (Matsuda & Tobari, 1989). Actually, mature spermatozoon has limited capacity to mount a DNA repair response and fully depends on the oocyte for protecting embryo development from paternally mediated genetic damage (Smith et al., 2013). In vivo implications of this research are related to recent studies that show the presence of melatonin in bovine follicular fluid (Gao et al., 2011), melatonin synthesis by bovine cumulus cells (El-Raey et al., 2011), melatonin receptors in oocyte and blastocyst embryos (Sampaio et al., 2012) and thereby a possible impact during in vivo fertilisation in these animals. Sperm capacitation and hyperactivation occur in the female reproductive tract, and these processes might be activated by melatonin from follicular fluid that is released into the oviduct after ovulation (Ishizuka et al., 2000; Casao et al., 2010). Taken together, adding 1 mmol melatonin to IVF media is deleterious for subsequent embryo development. Lower melatonin concentrations tested, despite their © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–12

Melatonin in bovine IVF and sperm function

modulation of sperm function (as antioxidant or pro-oxidant), do not improve in vitro embryo production in bovine. Further investigations are needed for determining whether melatonin can induce beneficial changes on embryo quality at molecular level. Acknowledgements The authors thank to local slaughter house (Frigor
ıfico Temuco) for supplying the ovaries. This study was supported by Grant FONDECYT 1100449 from CONICYT, Chile. Carolina Cheuquem
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