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Effect of the addition of α-tocopherol and ascorbic acid to the freezing medium on porcine semen traits

N Uc, F Centurión-Castro, E Reyes, J G Magaña-Monforte and J C Segura-Correa

Departamento de Reproducción y Mejoramiento Genético Animal, Universidad Autónoma de Yucatán, Mérida, Yucatán, México


The effect of α-tocopherol (Vitamin E), ascorbic acid (Vitamin C) and the combination of both, to the freezing medium on porcine semen traits was evaluated. The rich fraction of 30 ejaculates from six boars was used. The frozen ejaculates had at least 80% motility and 85% normal sperm. Each ejaculate was assigned to four treatments: T1 = 0.2 mg/ml of vitamin E; T2 = 0.45 mg/ ml of vitamin C; T3 = T1 + T2 and T4 = control. The vitamins were added to the semen during frozen, prior to their storage at 5ºC. The samples were frozen in liquid nitrogen and later thawed and evaluated at 37 ºC. The semen traits evaluated were: individual motility (MOT), mitochondrial activity (MA), acrosome integrity (AI) and plasma membrane integrity (MI). Data were analyzed by analysis of variance with repeated measures.

The addition of α-tocopherol (T1) improved (40.5%; p <0.001) MOT compared to the other treatments (T2 = 29.3%, T3 = 32%, and T4 = 26%, respectively). Likewise, α-tocopherol had a positive effect (p <0.05) on AI (46.4%) compared to the other treatments (38.4%, 37.9% and 36%). Ascorbic acid did not affect (p> 0.005) any of the semen traits, while the combination of both vitamins (T3) was different (p <0.05) only for MOT with respect to the control group (32% vs 26%). In conclusion, the addition of α-tocopherol to the freezing medium improved MOT and AI of frozen-thawed sperms, and the inclusion of both vitamins improved MOT compared to the control. The addition of ascorbic acid did not influence any of the semen traits.

Key words: semen motility, tropics, vitamin E, vitamin C


In recent years, research has been carried out to improve the fertilization capacity of frozen semen, since its use allows to maximize the benefits offered by artificial insemination; through, improving the reproductive performance of high genetic value boars, creating germplasm banks and favoring international trade for the import-export of semen doses (Johnson et al 2000; Hernández 2007). Despite the advantages of semen cryopreservation, the process itself causes alterations in sperm cells like any technique that includes their manipulation. In addition, boar sperms are sensitive to low temperatures, which cause various structural and functional alterations, related to oxidative stress.

Several methods have been proven to preserve the integrity of sperm membranes during freezing the semen. An important alternative to reduce or inhibit oxidative damage is the inclusion of compounds with antioxidant capacity to the freezing diluent, trying with this, to improve the viability and fertility of cryopreserved sperm (Rodríguez 1999).

α-Tocopherol (vitamin E) is a powerful antioxidant that captures free radicals in the plasma membrane, preventing the propagation of the chain reaction of lipid peroxidation (Almeida 2005). On the other hand, ascorbic acid (vitamin C) is an antioxidant that, due to its chemical structure, donates electrons to oxidized vitamin E (α-tocopherol); in this way, it restores and potentiates the antioxidant function of vitamin E (α-tocopherol), helping to protect the lipid membrane from peroxidation (Chihuailaf et al 2002; Sánchez and Mendoza 2003). Both antioxidants have been applied individually in the cryopreservation of semen of various species such as bovines and pigs, obtaining favorable results (Breininger et al 2005). The addition of antioxidants in the freezing medium can strengthen the antioxidant system of the sperm and consequently reduce oxidative stress and lipid peroxidation, preserving cell viability during freezing and thawing. Studies of the combined application of both antioxidants on the structural and functional characteristics of sperm cells are scarce. The objective of the present study was to evaluate the addition of α-tocopherol, ascorbic acid and the combination of both, to the freezing medium, on porcine semen traits.

Materials and methods


The study was carried out at the Faculty of Veterinary Medicine and Animal Science of the “Universidad Autónoma de Yucatán”, located at km 15.5 of the Merida-Xmatkuil road, Yucatan, Mexico. The climate of the region is warm subhumid with rains in summer (AW0). The average annual ambient temperature is 26.7 ºC, the relative humidity 80% and the average annual rainfall 1062 mm (INEGI 2010).


Six boars of the PIC line from 2 to 4 years old, located in different farms in Yucatan, were used, and they were under a similar feeding and management regime.

Collection and evaluation of semen

Semen was collected with the gloved hand method, using a fixed rack. Before mounting, the boar foreskin area was cleaned with disposable paper, and the content of the preputial diverticulum was removed. The semen obtained was deposited in a thermos provided with a disposable bag inside and a filter in the opening; using only the sperm-rich fraction; which, was distinguished by its milky coloration. Semen collections were carried out at intervals of 5-7 days and 5 ejaculates were obtained from each boar.

Once the sample was obtained, it was subjected to a water bath at 37 ºC and a general evaluation was carried out, which consisted in determining the volume and motility. The volume of the ejaculate was measured from a graduated cylinder and subsequently a 1:2 dilution was made with BTS (Beltsville Thawing Solution). For mass motility evaluation, a drop of concentrated semen was taken, placed on a slide, and observed with a bright field microscope using the 100x objective. Sperm mass motility was scored in crosses, according to the Zemjanis scale (1984). Subsequently, a drop of semen was deposited on a slide while placing a coverslip to observe individual motility (MOT), with an objective of 400x magnification. The type of movement was evaluated and the percentage of motile spermatozoa (0-100%) was scored after observing 3 or 4 fields of the smear.

After semen dilution with BTS, it was deposited in Falcon tubes inside a thermos and transported to the laboratory of reproduction of the Faculty of Veterinary Medicine and Animal Science to continue with the process. When the semen arrived at the laboratory, it was evaluated again to verify that the sample maintained adequate motility (not less than 80%); then the sample was deposited in 50 ml Falcon tubes to facilitate handling, and allowed to stand for 1 hour at laboratory temperature (24 ºC). During that time, the concentration of the ejaculate was evaluated. Semen samples were diluted 1:100, taken 0.1 ml of pure semen and placed in a tube with 99.9 ml of physiological saline solution with methylene blue and 1% formaldehyde. The Burker chamber was filled with the help of a Pasteur pipette, and spermatozoa were counted with the objective of 400x magnification, expressing the result as the number of sperm per milliliter.

To evaluate the sperm morphology, a solution with 1% formaldehyde and pure semen was made. A drop of the mixture was taken and the morphology was determined in a phase contrast microscope, with the objective of 1000x magnification and 100 spermatozoa were counted for each sample, and it was qualified as percentage of primary or secondary anomalies according to the place of affection, either the head, middle part or the tail (Zemjanis 1984).

Preparation of diluents

Once the total volume of the diluent to be prepared was known, the proportions for each LEY component were made, which corresponded to 20% egg yolk, 40 µl / 100 ml of kanamycin, 8.49 g/100 ml of β-lactose and 80% of distilled water. They were then added to a flask, which was placed on a magnetic stirrer to achieve better homogenization and subsequently kept at 16 ºC. The LEYGO was made up of 92.5% LEY, 6% glycerol and 1.5% Orvus ES paste, and was kept at a temperature of 5 °C.

Frozen semen

Ejaculates suitable for freezing had a minimum of 80% motility and 85% normal sperm (Roca et al 2006), and they were frozen using the method of Thurston et al. (1999). After one hour at laboratory temperature, the samples were transferred to a conservator, kept there for 3 hours at a temperature of 16 °C. Then, the packed semen was centrifuged at 800 x g for 15 minutes in a refrigerated centrifuge (Hermle, Germany). Next, the samples were removed from the centrifuge and transported to a cold room at 16 ºC. The supernatant was removed from each tube with the help of a plastic pipette, and the resulting semen pellet was added with the LEY diluent and stirred to achieve adequate homogenization. The tube with the semen was placed in a glass with water at 16 ºC and placed in another refrigerator with a temperature of 5 °C, where it remained for 2 hours. The introduction of the tube with the semen in water was carried out with the purpose of avoiding a sudden change in temperature and that the decrease of temperature was gradual. After this period, the semen was returned to the cold room to add the LEYGO (Lactose, egg yolk, glycerol, Orvus ES paste).

The amount of LEY and LEYGO added depended on the initial concentration of the ejaculate. The inclusion of diluents served to adjust the initial concentration to 1500 x 106 spermatozoa/ ml and the final concentration to 1000 x 106 sperm/ml. The straws were then filled with semen using a packing machine (Foruard, France). Each straw contained 500 x 106 sperm. Subsequently, the sealed straws were placed on a steel base and placed in a polystyrene box, at a distance of 5 cm from the surface of the nitrogen. The straws remained there for 20 minutes, and then they were directly immersed in liquid nitrogen. The frozen straws were placed in the nitrogen thermos, where they remained until thawed.

Experimental treatments

Each ejaculate was divided into four fractions before adding the LEY diluent. After the corresponding amount of LEY was added to each tube, the evaluated antioxidants were added. The treatments were T1 = 0.2 mg/ml of vitamin E; T2 = 0.45 mg/ml of vitamin C; T3 = T1 + T2; T4 = control. Once the LEY and the antioxidants were added, the semen continued with the freezing process.


Four straws per treatment were thawed 5 days after frozen. Each straw was removed from the thermos with nitrogen and placed in a water bath at 37 °C. The straw was vigorously shaken for 20 seconds. Subsequently, the excess water was wiped off with paper and the sealed end of the straw was cut to deposit the semen in a tube kept in the water bath. A 1: 2 dilution with BTS was made and one minute was waited for its evaluation.

Viability of thawed semen

Individual motility (MOT) was evaluated by subjective analysis based on direct observation of the seminal sample through the microscope, establishing the percentage of cells with rectilinear movement. The acrosome integrity (AI) of the sperm was determined using a phase contrast microscope at 1000x magnification, in which the sperm were deposited in a formaldehyde solution prior to evaluation. Normal acrosomes were those that presented a dark, sharp and well-defined crescent shape (Centurión 2002).

Finally, the membrane integrity (MI) and mitochondrial activity (MA) were analyzed using the fluorescence technique using carboxyfluorescein diacetate (DCF) and Rhodamine 123. Two straws from each treatment were mixed and 500 µl of semen were taken. The sample was placed in a dark room and 20 µl DCF and 6 µl rhodamine were added; then, it was incubated in an oven at 37 ºC for 10 minutes. After that time, 4 µl of the sample was taken and observed in an epifluorecent microscope (Olympus, Japan) at 1000x magnification. First, 100 spermatozoa were counted to determine the integrity of the plasma membrane, considering as integral cells the spermatozoa that showed a green coloration on the head. Subsequently, the same volume was taken from the same sample and the mitochondrial activity was analyzed, considering as active spermatozoa those that showed a green fluorescence emission in the intermediate part (Nagy et al 2003), the result for both response variables was expressed as percentage.

Statistical analysis

Data were analyzed by a completely randomized design with repeated measures using a commercial program (SAS 2000). The experimental unit was the ejaculate and the repeated measurements, each of the four fractions of the ejaculate. The response variables were the individual motility (%), spermatozoa with mitochondrial activity (%) and spermatozoa with membrane and integral acrosome (%). The means ± standard error were considered different when the probability of a type I error was less than 0.05 (P <0.05).


The results by treatment for MOT and AI of frozen-thawed semen are shown in Table 1. The effect of α-tocopherol (Vitamin E) was important (p <0.001) since it was the treatment with the highest percentage of mobile cells and intact acrosomes. Regarding ascorbic acid (Vitamin C), the means were similar for all the characteristics studied, without differences between treatments (p> 0.05). Regarding the effect of vitamin E + C on MOT, a difference was only observed for the control group (p <0.05). In addition, no difference (p> 0.05) of the group of vitamin E + C on AI compared to the other treatments, and no treatment effect on MI and MA (p> 0.05) were found.

Table 1. Means and standard errors by treatment on some frozen-thawed semen traits of boars


MOT (%)

AI (%)



Vitamin E

40.5a ± 1.3

46.4 a ±2.5

58.8 a ± 2.4

50.7 a ± 2.4

Vitamin C

29.3bc± 1.4

38.4 b± 2.9

58.1 a ± 2.7

49.8 a ± 2.7

Vitamin E+C

32.0b± 1.3

37.9 b± 2.6

59.3 a ± 2.5

49.5 a ± 2.5


26.0c± 1.3

36.0 b± 2.5

56.3 a ± 2.4

45.8 a ± 2.4

MOT= Individual motility; AI= Acrosome integrity; MI= Plasmatic membrane integrity; MA= Mitochondrial activity


The frozen- thawed process has been reported to primarily damage sperm motility. The results in this study show a significant effect (p <0.001) of α-tocopherol on the motility (%) of thawed sperm. The effect of α-tocopherol on sperm motility agree to that by Breininger et al (2005) and Satorre et al (2007), who observed an improvement in the percentage of MOT in the samples treated with vitamin E compared to the control group. However, MOT in the present study was higher than that reported by Breininger et al (2005), 40.5 vs 37.0%, and lower than those obtained by Satorre et al (2007), 46% vs 40.5%. The results obtained here also agree with those by Peña et al (2003), who reported an improvement in thawed sperm motility using an α-tocopherol analog. A low motility is associated with membrane fragility and spermatozoa with abnormalities in the intermediate piece of the spermatozoa, which affect the anti-peroxidation systems; therefore, the peroxidation processes induce a rapid and irreversible loss of motility. α-tocopherol has been shown to inhibit lipid peroxidation (Satorre et al 2007).

AI is one of the main sperm traits in evaluating sperm viability and membrane functionality (Garner and Johnson 1995). The best results obtained in this study with respect to intact acrosomes, adding α-tocopherol, showed a trend similar to that obtained by Satorre et al (2007), who used the same concentration of vitamin E. These studies coincide with those carried out with bovine semen by Beconi et al (1993) suggesting that the addition of α-tocopherol to the medium cause a reduction in the proportion of spermatozoa with acrosome reaction, since it decreased the effect of reactive oxygen species (ROS), which affect acrosome integrity, causing a premature capacitance in sperm.

The higher percentages of MOT and AI of spermatozoa after thawing with α-tocopherol could be explained by the antioxidant capacity of this vitamin; since it protects the sperm from the damage caused by oxidative stress associated with the frozen process (Torres et al 2002; Breininger et al 2005), thus contributing to maintaining the sperm viability essential for fertilization. On the other hand, contrary to what was expected, the results of this study did not show significant differences among treatments on MI and MA; however, this lack of difference agree with Satorre et al (2007), who also did not find a protective effect on these structures. A greater effect of α-tocopherol on membrane integrity was expected, since there is evidence that α-tocopherol act on the membrane lipids, protecting it from the action of free radicals.

Regarding ascorbic acid, this study did not show an effect on sperm quality traits. This agrees with Sönmez and Demirci (2004) who worked with semen from rams, and found no effect on MOT and AI in thawed semen, using different concentrations of ascorbic acid (0.5, 1 and 2 mg / ml). Similar results found Hernández et al (2003) with thawed bovine semen adding 0.2 mg/ml and 0.4 mg/ml of vitamin C. On the other hand, Yashimoto et al (2008) using frozen-thawed semen from Okinawan native pig found that the addition of a supplement with 200 μM AA-2G of ascorbic acid 2- 0- α glucoside (AA- 2G), had the most beneficial effect on sperm motility and cell membrane integrity of spermatozoa upon thawing.

Although studies in horses on the effect of ascorbic acid in chilled semen (Aurich et al 1997) have shown effects on MI and MOT in semen kept at 5 °C, using 0.45 and 0.9 mg / ml of ascorbic acid, it not clear why no effects have been found with the addition of vitamin C to the freezing medium. These results suggest that under the conditions of this study, the addition of ascorbic acid does not have any inhibitory effect on oxidative stress in thawed boar sperm. Therefore, the lack of effect of the treatments is difficult to explain, although a greater susceptibility of boar sperm cells to thawing is not ruled out compared to sperm from other species including sheep and bovine sperm cells (Satorre et al 2007). Probably, the nature and/or concentration of ascorbic acid used have influenced on the results. However, although no improvement due to the addition to the freezing medium was observed, a detrimental effect on any of the evaluated traits was not found. Therefore, a toxic effect of the ascorbic acid concentrations used in this study could be discarded (Sönmez and Demirci 2004).

The addition of α-tocopherol and ascorbic acid to the freezing medium had a positive effect on sperm motility compared to the control. Unfortunately, there are no studies evaluating the effect of both vitamins on semen viability. However, different authors establish that there is a synergistic reaction between ascorbic acid and α-tocopherol; manifesting itself in a restoration of the antioxidant activity of α-tocopherol by ascorbic acid (Chihuailaf et al 2002; Sánchez and Mendoza 2003). Here, it was observed that it was possible to counteract to some extent the harmful effects of free radicals formed during the freezing process that affect the cellular structures involved in cell movement (Johnson and et al 1980).



The study was funded by Fundación Produce Yucatán, AC.


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