Effect of different cryopreservation extenders added with antioxidants on semen quality and in vitro embryo production effi ciency in cattle

To evaluate the addition of antioxidants in extenders on post-thaw bovine semen quality and in vitro embryo production effi ciency. Six semen samples were collected from fi ve Holstein bulls. In the experiment I, the samples were diluted with AndroMed® and Bovimix® and added antioxidants glutathione (1.5 and 2.5 mM) and melatonin (0.5 and 1.0 mM). In the experiment II, the best treatments obtained in experiment I were used for in vitro fecundation. Glutathione did not improve sperm viability. Melatonin had a negative effect on semen characteristics. Andromed® showed better results in sperm kinetics parameters. Bovimix® was more effi cient in maintaining cell integrity parameters. Signifi cant correlation was found between sperm kinetics parameters and between cell integrity parameters. For in vitro embryo production, after oocyte selection, maturation, fertilization and cultivation were performed using the four treatments previously evaluated. Andromed® was more effi cient in the cleavage rate, no effect of the addition of glutathione. However, the addition of 2.5 mM glutathione in the Bovimix® improved the cleavage rate. There was a signifi cant moderate correlation between cleavage rate and sperm kinetic characteristics. Glutathione did not improve sperm viability. Melatonin reduced the maintenance of sperm characteristics. Andromed® was more effi cient in in vitro embryo production and no effect of glutathione was found in this extender. Addition of 2.5 mM glutathione in the Bovimix® extender provided a higher cleavage rate.


INTRODUCTION
Semen quality is of utmost importance for the success of cattle artificial insemination programs. Thus, it is essential to maintain the characteristics of sperm through an adequate cryopreservation process (Schober et al. 2007), which includes the use of appropriate extenders and the addition of antioxidants to prevent the excessive production of reactive oxygen species (ROS) (Raheja et al. 2018).
Egg yolk is one of the most used substances for freezing bovine semen. However, its use is discussed because it contains constituents that inhibit sperm cell respiration and may lead to reduced motility, and the great risk of microbiological contamination, as it is an animal product ( Crespilho et al. 2012). As a consequence, the World Organization for Animal Health (OIE), recommended in the 2003 Terrestrial Animal Health Code, that animal origin products used in semen processing should be free of any biological risk or processed in order to ensure the safety of such compounds (Marco-Jiménez et al. 2004). As a result, substitution of egg yolk by products of plant origins, such as soybean lecithin in cryopreservation extenders, has been currently evaluated for the dilution of bull semen and it was shown efficient in conserving post-thaw motility and fertility (Murphy et al. 2018).
Antioxidant substances have also been added to the extender media to minimize the deleterious effects caused by ROS on sperm of various species, including cattle. Antioxidants, such as glutathione and melatonin, slow the oxidation rate by maintaining ROS production of at physiological levels (Ashok et al. 2014), resulting in high quality semen samples and consequent increase in the number of in vitro produced embryos (Pang et al. 2016). Shah et al. (2017) indicated that glutathione at 0.5 mM can be effectively used as an additive in bull semen egg yolk tris glycerol extender for freezing and thawing, to protect the sperm from deleterious effects of cryopreservation. Other study found that the addition of 1 mM glutathione in the lecithin-based extender provided better quality to bovine semen and better in vitro fertility (Mousavi et al. 2019).
Supplementation of 2 or 3 mM concentration of melatonin in the bull semen extender improved the quality of post-thawed bull semen (Ashrafi et al. 2013). Pang et al. (2016) concluded that 0.01 mM and 1 mM melatonin improved sperm quality but only 1 mM increased blastocyst development rate of in vitro-produced bovine embryos.
In this context, this study aimed to evaluate the effect of extenders based on egg yolk (Bovimix ® ) and soybean lecithin (Andromed ® ), and the addition of glutathione (1.5 and 2.5 mM) and melatonin (0.5 and 1.0 mM) on the quality of post-thawed bovine semen. In addition, checking the fertility of these samples on the bovine in vitro embryo production efficiency and the correlation between sperm characteristics with in vitro embryo production (IVEP).

Animals, semen collection and evaluation
The study was conducted at the Animal Reproduction Laboratory of the Instituto Federal Goiano, Rio Verde, Brazil, after approval by the Ethics Committee of the same institution, protocol 011/2014.
Five Holstein bulls were selected by andrological examination. A total of 30 ejaculates (six ejaculates/bull) were collected from bulls using electroejaculator (Boijector ® , model 65A). For collection, the animals were kept in a containment trunk and the prepuces were cleaned with paper towels. After collection, the percentages of total motility (%), progressive motility (%) and vigor (0-5) were evaluated by optical microscopy of a semen drop between slide and coverslip preheated to 37°C. Only the ejaculates that met the criteria recommended by the andrological examination and semen evaluation manual of the Colégio Brasileiro de Reprodução Animal (Brasil 2009), were used in the experiment.

Semen freezing
The samples were homogenized, stored in 0.5 mL plastic straws and sealed, placed on a stainless steel tray and refrigerated in a Minitub ® refrigerator (Porto Alegre, State of Rio Grande do Sul) at 5°C for five hours. The trays were then placed 3 cm above the nitrogen level in a 40 L Styrofoam box. After 15 minutes in nitrogen vapor, straws were immersed in liquid nitrogen, racked and stored in cryogenic device for further analysis.

Thawing of semen
The straws cryopreserved were removed from liquid nitrogen and thawed in water bath at 37˚C for 30 seconds. This procedure was used for all sperm evaluations.

Sperm kinetics analysis
Sperm kinetics analyses of semen samples were performed by the CASA software (Sperm Analysis System, Ivos Ultimate 12's, Hamilton Thorne Bioscences, Beverly, MA, USA), previously adjusted for bovine sperm analysis. An aliquot 10 µL of thawed semen sample (1,2x10 6 motile sperm) was mounted on the reading slide (Makler, Santa Ana, CA, USA) heated at 37ºC. Then three fields were selected for reading and analysis of the samples.

Flow Cytometry Assessment
Membrane integrity, acrosomal integrity, and membrane stability were evaluated according to the manufacturer's recommendations for analysis of bovine semen in a flow cytometer (FlowSight ® -Amnis Imaging Flow Cytometers, Merck Milipore, Darmstadt, Germany). An aliquot 10 µL of thawed semen sample (1,2x10 6 motile sperm) was incubated at 37°C for 10 minutes in a conical microtube in a fluorescent probe solution according to the analysis, protected from light. For each thawed sample, were performed membrane integrity, acrosomal integrity and membrane stability analyzes.

Membrane integrity analysis
Membrane integrity analyses were determined by combining SBER-14 fluorescent probes (100 nM/mL) and propidium iodide (2 mg/mL, Sperm Viability Kit L-7011, Molecular Probes Inc, Eugene, OR, USA), to check intact and damaged cells.

Experiment II -in vitro embryo production (IVEP)
Six replicates of IVEP were performed with four best treatments obtained in Experiment I. To avoid bull effect, for in vitro fertilization, the best bull was selected according to in vitro analyzes of post-thaw semen by CASA method and flow cytometer obtained in Experiment I.
We evaluated the cleavage rate three days (D3) after IVF, blastocyst rate seven days (D7) after IVF and hatching rate eight (D8) and nine (D9) days after IVF. The blastocyst rate was calculated on the cleavage rate, and the hatching rates calculated on the blastocyst rate.

Oocyte selection and in vitro maturation
Crossbred cow ovaries from animals of the slaughterhouse were collected and transported in thermos containing physiological saline solution (0.9% NaCL) at 35°C to the laboratory and processed within 4 hours.
In the laboratory, the ovaries obtained were washed with the same solution and the cumulus-oocyte complexes (COC, n=1983, six replicates per treatment) aspirated from 2 to 8 mm diameter follicles with the aid of 10 mL syringes and 40x12 mm needles. Soon after, COCs were stored in 15 mL conical tubes and kept in a water bath at 35°C for 10 minutes for decantation.
In a conical microtube, 95 μL distilled water was added with 5 μL semen resuspended in FEC medium for counting sperm concentration in a Neubauer chamber. Immediately afterwards, the inseminating dose to be added to the fertilization drops was calculated to give a final concentration of 1x10 6 SPTZ/mL.

In vitro embryo cultivation
Sperm and oocytes were incubated for 10 hours at 38.8°C in 5% CO 2 and saturated relative humidity without condensation. In vitro insemination day was considered as day zero (D0). Zygotes from fertilized oocytes were washed and transferred to 200 μL droplets of culture medium (modified synthetic oviduct fluid (Holm et al. 1999) supplemented with 2.7 mM myo-inositol, 0.2 mM pyruvate, 2.5% foetal bovine serum (v/v), 5 mg/mL BSA-fatty acidfree and 75 μg/ mL amikacin) and incubated at 38.8 °C in 5% CO 2 for seven days. The cleavage rate was evaluated 72 hours after insemination (D3), and half of the culture medium of each drop was replaced. Structures were considered cleaved when the cellular division with at least 2-4 cells was present. The morphological quality of the embryos was measured according to International Embryo Transfer Society (IETS) guidelines (Bó and Mapletoft 2013). According to IETS, the quality of the embryos is classified in Grade 1 to 4 (1-excellent/good, 2-regular, 3-poor, 4-unviable). Embryos are graded regarding the number of physical characteristics including shape, colour and density of cytoplasm or inner cell mass, number and compactness of cells, area of perivitelline space, number of extruded or degenerate cells and frequency and size of cytoplasmic vacuoles. The blastocyst stage was observed seven days (D7) after IVF. Hatching rates were assessed eight (D8) and nine (D9) days after IVF.

Statistical analysis
Data analyzed were randomly distributed in blocks, with the bull being the block. As for the analysis of the characteristics of sperm kinetics, different antioxidants and extenders evaluated, data were log transformed, because the data did not have normal distribution, and subsequently tested by nonparametric Friedman's test (P<0.05).
To compare the percentage of cleavage rate, blastocysts and hatching between treatments, the Chi-square test was applied.
It was also estimated the correlation between the variables of in vitro semen quality and the efficiency of in vitro embryo production, seeking to evaluate the association between characteristics, using the Spearman correlation coefficient (r).
For the interpretation of the correlation coefficients, values of r between 0.90 and 0.70 were considered as high magnitude correlations, between 0.69 and 0.40, moderate correlations, between 0.39 and 0.20, low correlations, and between 0.19 and 0.00, weak correlations (Silva 2011).
The analyses were run using the R Project for Statistical Computing program through R Project version 3.2.2 (R Core Team 2015).

Experiment I
Andromed ® extender without the addition of glutathione at different concentrations was found to improve sperm kinetic characteristics of bovine semen. Melatonin reduced semen viability. Bovimix ® extender obtained better results in terms of cell integrity.
The total motility percentage in the AG 1.5 treatment was equal to the AC treatment and higher than the other treatments. Progressive motility was similar between the AC, AG 1.5 and AG 2.5 treatments, but higher than the other treatments (Table I). These results are similar to those observed for the variables VAP, VSL, VCL and ALH.
Regarding the use of different extenders, a difference was found in both treatments in relation to sperm kinetic characteristics. Andromed ® extender showed better results in maintaining sperm kinetic characteristics. However, Bovimix ® extender obtained better results regarding the membrane integrity (IM), acrosomal integrity (PNA) and membrane stability (EST), as presented in Tables I and II.
Considering the addition of melatonin, it can be observed that in the two concentrations tested, melatonin reduced the maintenance of sperm viability after freezing, as observed in membrane integrity (IM) and acrosomal integrity (PNA). This is because the concentration used is toxic to sperm, which reduced their viability. Table III lists the correlations between the semen quality characteristics evaluated. High and significant positive correlations were found between MT and MP, VAP and VCL, between MP and VSL, VSL and ALH, STR and LIN, IM and PNA, IM and EST, and PNA and EST.
Moderate and significant positive correlation estimates were observed between MT and VAP, VSL, VCL and ALH. As well as between MP and the characteristics VAP, ALH, and BCF. Moderate and significant positive correlation estimates were also verified between VSL and BCF, ALH and BCF, ALH and STR parameters.  The correlations between sperm kinetic characteristics and cell membrane integrity were significant, with low magnitude.

Experiment II
Based on the best in vitro analyzes of post-thaw semen by CASA method and flow cytometer obtained in Experiment I, the groups BC, BC2.5, BG and BG2.5 were selected for in vitro fertilization.
Regarding the in vitro embryo production, the effect of using different cryopreservation extenders on the cleavage rate was observed, with a difference between the treatment with Table III soybean lecithin (Andromed ® ) and egg yolk (Bovimix ® ). Soybean lecithin-based extender showed higher cleavage rate, but without difference between treatments for blastocyst rate and hatching rate on days eight (D8) and nine (D9) after IVF (Table IV). The addition of glutathione in soybean lecithin-based cryopreservation medium (Andromed ® ) did not provide higher cleavage rates when compared to Andromed ® treatment without this antioxidant. However, the addition of 2.5 mM glutathione in the egg yolkbased medium (Bovimix ® ) for bovine sperm cryopreservation improved the cleavage rate when compared to Bovimix ® treatment without glutathione.
Regarding blastocyst and hatching rates on day eight (D8) and nine (D9) after IVF, no difference was detected in the evaluated treatments. Table V presents the correlation values between sperm kinetic parameters of bovine semen and cell integrity analyzed by a cytometer, as well as cleavage rate, blastocyst and hatching rates on day eight (D8) and nine (D9) after IVF.
There was a significant moderate positive correlation between the characteristics of velocity (VAP, VCL and ALH) and the cleavage rate. The variables LIN, IM and PNA showed significant moderate negative correlations with the cleavage rate. Regarding blastocyst and hatching rates on day eight (D8) and nine (D9) after IVF, no correlation was detected with the evaluated sperm characteristics.

DISCUSSION
Our findings suggest further research on the appropriate concentration of antioxidants to be used to combat the deleterious effects of reactive oxygen species and improve sperm quality. The concentration used may cause reduction in semen viability due to the higher cytotoxic effect caused by the high concentration, mainly melatonin. As for extenders, Andromed ® showed better results in maintaining the kinetic characteristics of bovine semen, regardless of the addition of antioxidants, and can be used instead of egg yolk-based extender (Bovimix ® ).
In this research, melatonin reduced sperm cell viability at the evaluated concentrations (0.5 and 1.0 mM) and in the different extenders tested, negatively influencing the sperm quality of the samples. It is believed that melatonin at Table IV. Percentage values of cleavage rate (TC), blastocyst rate (TB) and hatching rate on day eight (TE -D8) and nine (TE -D9) after fertilization with cryopreserved Holstein semen with extender based on soybean lecithin (AndroMed ® ) and egg yolk (Bovimix ® ), with and without the addition of glutathione at different concentrations.

Groups
Number the evaluated concentrations may have caused cytotoxic effect on sperm cells. Nevertheless, when 0.01 mM and 1 mM melatonin was added in thawed bovine semen and incubated for only 1 h at 38.5˚C in humidified air with 5% CO 2 before in vitro fertilization, led to higher plasma membrane integrity, mitochondrial activity and acrosome integrity, and significantly decreased intracellular ROS levels; increases the blastocyst rate and it decreases apoptosis rate (Pang et al. 2016). This protective effects of this antioxidant on spermatozoa are associated with a reduction in lipid peroxidation as a consequence of increasing the total antioxidant capacity and antioxidant enzymes activity like catalase (Ashrafi et al. 2013). According ChaithraShree et al. (2019), the addition of 0.1 mM melatonin protected the plasma membrane and acrosome region and maintained the ultrastructure integrity of the cryopreserved bovine spermatozoa when compared to control group, whereas the electron micrography of spermatozoa treated with 0.2 and 0.25 mM melatonin illustrated highest damage to the plasma and acrosome membrane.  It may be that the negative results presented by melatonin´s groups in this study were due to toxicity of the solvent, either by type or concentration or by not being included in the control group. The melatonin stock solution was based on ethanol, obtaining a final concentration of 2% in the samples. Pang et al. (2016), the final concentration of ethanol used was 0.01% and they were not used in the control because obtained the same results without this solvent. Ashsafi et al. (2013)  Thus, addition of melatonin during sperm freezing at the concentrations used during the present study did not maintain the sperm characteristics. However, its beneficial effects appear to depend on the solvent used in the preparation of the stock solution, melatonin concentration and timing of its use.
No effect of glutathione was observed on ovine sperm characteristics cryopreserved using tris-egg yolk extender supplemented with 0, 1, 5 and 10 mM glutathione (Perez et al. 2012) nor with 100, 200, and400 mM (Câmara et al. 2011). On the other hand, Mata-Campuzano et al. (2014) observed that 5 mM glutathione had a positive impact on ovine semen quality because improved mitochondrial activity compared with 0.2 and 1 mM, but did not see increase in farrow rates sheep after artificial insemination. However, addition of 1 mM glutathione in the lecithin-based extender provided better quality to bovine semen compared with 0.5, 2 and 3 mM, promoting increase blastocyst and hatching rate at in vitro produced embryo (Mousavi et al. 2019), a result contrary to that observed in this study.
Thereby, adding glutathione at the correct concentration in bull semen preservation, maintain of the membrane integrity, important for sperm metabolism, motility, capacitation, acrosome reaction and the binding of spermatozoa to the egg surface (Yadav et al. 2019).
Due to the possible microbiological contamination caused by extenders based on animal products, Andromed ® has been widely used in cryopreservation of bovine semen. In this study, better results were observed regarding sperm kinetic characteristics when using soybean lecithin-based extender. Similar result was reported by Aires et al. (2003) who found greater motility in semen of bulls cryopreserved with soybean lecithin-based extender (AndroMed®)" compared with the egg yolk-based extender (73.54% and 59.57%, respectively). In turn, Salmani et al. (2014) evaluated different concentrations of soybean lecithin (0.5, 1, 1.5, 2 and 2.5%) and egg yolk (15%) in goat semen cryopreservation and found no difference in the characteristics of sperm kinetics and membrane integrity of semen diluted with both extenders.
Samples of sperm with high velocity, as observed with Andromed ® (VAP, VCL, VSL, and ALG) and low percentage of STR and LIN are indicative of cell hyperactivation, not by the type of antioxidant used but by the extender evaluated. Thus, this feature is considered undesirable since sperm activation should occur near the fertilization site. Thus, this early activation results in shorter sperm life span, reducing the fecundation capacity of the animal (Verstegen et al. 2002). Therefore, it is believed that Bovimix ® extender was more efficient in maintaining sperm characteristics, as it provided better percentages of membrane integrity and acrosomal integrity with lower velocities.
Thus, it is assumed that there was hyperactivation of cryopreserved cells with soybean lecithin-based extender, since higher mean values of velocity and low percentage of