Glutathione and IGF-1 in bovine seminal cryopreservation: oxidative stress response and pregnancy rate

The objective of this study was to evaluate the rate of conception, metabolic, and structural conditions of cryopreserved bovine sperm cells, plus extender with IGF-1 and glutathione (GSH). 12 ejaculations of Nelore bulls were used, submitted to treatments: control, gSH (2mM/mL), IGF-1 (100ng/mL) and gSH (1mM/mL) + IGF-1 (50ng/mL). After cryopreservation and thawing the semen passed the fast thermo resistance test (TTR), plasma membrane and acrosomal integrity (PIAI), mitochondrial membrane potential (AP), oxidative stress, and conception rate. Tukey test was used for the statistical analysis of the parametric variables and the Friedman test for nonparametric. The gestation percentage was compared by the Chi-square test. There was no statistical difference (P<0.05) between treatments for the TTRr variable. Otherwise in the oxidative stress evaluated with the CellROX probe was noted that the IGF-1 showed the highest number of reactive cells (P<0.05). The PIAI, AP and gestation rate showed no difference among treatments (P>0.05), with an average of conceptions of 36.58%. It is concluded that IGF-1, gSH and their association did not cause changes in sperm motility, mitochondrial potential, plasma and acrosomal membrane integrity. IGF-1 increased oxidative stress, however, there was no difference in the gestation rate among the treatments.


INTRODUCTION
Seminal plasma is a complex mixture of secretions originated in the testicles, epididymides, and accessory sexual glands (Manjunath et al., 1993), serving as a vehicle for ejaculated sperm, in addition to containing factors that influence their fertilization capacity (Calvete et al., 1996). Because of the interaction that exists between the seminal fluid and the sperm cells, the most recent research points to certain growth factors as determinants in the seminal quality. Among these, the insulin-like growth factor 1 (IGF-I) is considered one of the most important, as it favors the development of germ cells and the maturation of sperm (Skinner, 1991).
According to the review by Kumar et al. (2019), IGF-I supplementation improves semen quality in several species, such as dogs, stallions, and bulls, and their protective function may be due to antioxidant activities (Selvaraju et al., 2016), which is diminished or lost in the process of handling seminal plasma that routinely require dilution, reducing the concentration of these, which results in cell stress (Bilodeau et al., 2000). Another antioxidant that is lost in this seminal dilution is glutathione (GSH) which is responsible for the reconstruction of thiol groups (-SH) in proteins, which can be eliminated during oxidative stress, as well as protecting cell membranes from lipid oxidation preventing the formation of free oxygen (Lenzi et al., 1994). A great number of studies propose that the production of reactive oxygen species (ROS) and the semen's oxidative capacity enhance the harmful effects of seminal manipulation (Bustamante Filho et al., 2006).
Oxidative stress resulting from the imbalance between the concentration of antioxidants about the concentration of free radicals, generally (Nichi et al., 2006) imposes conditions that are extremely unfavorable to the maintenance of their viability. In spermatozoa, specifically, ROS causes structural damage to the acrosome, the head, and the intermediate part, as well as initiate apoptosis and induce DNA fragmentation (Castro, 2010). Knowledge about the oxidative damage caused to sperm cells resulting from the imbalance between the physiological concentrations of oxidants and antioxidants, resulting from increased production of ROS or reduced total antioxidant capacity of semen, can provide important information to improve sperm viability, mainly in post cryopreserved samples (Guerra et al., 2004). Thus, to improve the preservation of cell integrity, the association of antioxidants and hormones has been used in the semen freezing extenders of several species, including bovine (Beconi et al., 1993). Therefore, the objective of this study was to evaluate the conception rate, the metabolic and structural conditions of bovine sperm after freezing, using diluents supplemented with IGF-1, glutathione, and its association.

MATERIAL AND METHODS
The present study was approved by the Ethics committee of the Federal University of Acre, under the number 049/2015. The steps of cryopreservation of semen and artificial inseminations were performed in the municipality of Grajaú/MA, located 05°49'08" S and 46°08'20" O, with a tropical climate (Aw). The experiment was conducted at the State University of Maranhão, College São Luís/MA, and at the School of Veterinary Medicine and Animal Science (FMVZ) of University of São Paulo (USP), College Pirassununga/SP. Six Nellore bulls were used with average weight and age of 500kg and 36 months, respectively, classified as apt for reproduction, according to the Brazilian College of Animal Reproduction (Henry and Neves, 2003). All of them were collected by electroejaculation, going through repetition with an interval of one month, totalizing 12 ejaculation, which were divided among the treatments, evaluating volume, concentration, motility, vigor, and spermatic morphology.
For the post-thawing analysis, two semen straw per treatment was used, thawed in a water bath at 37°c for 30 seconds, evaluating motility and vigor. For the rapid thermoresistance Test (TTRr), a seminal aliquot was maintained at 45°c for 30 minutes, spermatic motility and vigor were evaluated every 10 minutes, from the moment 0. The morphology of the spermatic cells was performed according to Henry and Neves (2003), with the count of 200 cells through the technique of moist preparation under differential interference contrast microscopy (DIC), being classified into major and minor defects, according to Blom (1972).
The reactive oxygen species (ROS) were evaluated using the CellROX Fluorescent Probe™ Deep Red reagent, using 1µL de H3342 e 2µL de CellROX TM , incubated for 30 minutes at 37°c and the reading performed under epifluorescence microscopy (microscope of epifluorescence mark Nikon, model Eclipse 80i), classifying them with or without oxidative stress (Alves et al., 2015). Semen fertility was performed with 60 heifers per treatment, totalizing 240 animals inseminated at the fixed time (TAI) with the three-pass protocol (D0 -P4 implant and 2mg of estradiol benzoate; D8withdrawal of P4, 0.52mg of PGF2α, 1mg of estradiol cypionate and 300 IU of eCG; D10 -0.01mggnRH). After 45 days of insemination, the pregnancy diagnosis was performed using ultrasound evaluation using a transrectal linear probe with a frequency of 5 MHz (Mindray DP 2200Vet). For statistical analysis, the variables went through the normality tests of Shapiro-Wilk and Lilliefors, and the parametric parameters (PIintact plasma membrane; AI -intact acrosome; PIAI -spermatozoa with intact plasma and acrosomal membranes; AP -high mitochondrial potential and oxidative stress) were evaluated by ANOVA, comparing the averages through the Tukey test and the non-parametric (Motility, vigor, and TTRr) by Friedman's test, with a significance of 5%. The percentage of pregnancy was compared utilizing the chi-square test.

RESULTS
After cryopreservation, when motility, vigor, major and minor defects were evaluated, no significant difference was observed (P>0.05) between treatments (Table 1). There was no difference between treatments (P> 0.05) by the Friedman test. gSH:glutathione; IGF-1: insulin-like growth factor 1; gSH + IGF-1: association of glutathione with insulin-like growth factor 1.
These values were adequate for insemination since they had at least 37% motility (control group) after thawing, being above the minimum required, 30%, by the Brazilian College of Animal Reproduction (Henry and Neves, 2003). The major (%) and minor (%) defects found in fresh semen and the control group were 5.5 ± 1.16, 3.25 ± 1.28, 7.29 ± 1.81, and 3.45 ± 1.68, respectively, with no significant difference between the evaluations before and after the seminal freezing. By the evaluation through the rapid Thermoresistance Test (TTR) there was no significant difference between the treatments (P>0.05). There is a difference (P<0.05) only between the initial and final values (TTR) within each treatment, but not between them (Table 2).
When evaluating the percentages of cells with mitochondrial potential (AP), plasma membrane (PI), and acrosomal membrane (AI) demonstrated by the association of fluorescent probes, it was not possible to verify a significant difference (P>0.05) between the treatments (Table 3). When analyzing the oxidative stress with the aid of the CellRox TM Deep Red probe, responsible for evaluating the amount of ROS, such as hydroxide and hydrogen peroxide, present in the sperm cells, with and without oxidative stress, it was observed that IGF-1 presented higher (P<0.05) number of cells with oxidative stress when compared with the other treatments (Table 4). It was observed that the pregnancy rate per treatment did not differ (P>0.05), with the mean and standard deviation between treatments of 36.58 ± 2.51% (Table 5). Table 2. Mean and standard deviation (X ± S.D.) of motility at time 0 ', 10 ', 20 ', and 30' of thermoresistance test in different treatments of cryopreserved semen of Nellore bulls Different letters in the same column differ (P <0.05) by the Friedman test. gSH:glutathione; IGF-1: insulin-like growth factor 1; gSH + IGF-1: association of glutathione with insulin-like growth factor 1. ∆: Difference between initial and final values. Table 3. Mean and standard deviation (X ± S.D.) of spermatozoa with acrosomal e plasma membrane intact and mitochondrial potential in different treatments of cryopreserved semen of Nellore bulls There was no difference between treatments (P> 0.05) using the Tukey test. PI -intact plasma membrane; AI -intact acrosome; PIAI -spermatozoa with intact plasma and acrosomal membranes; AP -high mitochondrial potential. Table 4. Mean and standard deviation (X ± S.D.) of spermatozoa with oxidative stress in different treatments of cryopreserved semen of Nellore bulls Different letters on the same line differ by Fisher's exact test (P <0.05). gSH:glutathione; IGF-1: insulin-like growth factor 1;gSH + IGF-1: association of glutathione with insulin-like growth factor 1. Table 5. Percentage of pregnant Nellore heifers after insemination with different treatments of cryopreserved semen of Nellore bulls GSH:glutathione; IGF-1: insulin-like growth factor 1;gSH + IGF-1: association of glutathione with insulin-like growth factor 1.

DISCUSSION
The addition of gSH was expected to provide greater spermatic motility and vigor than the other treatments since antioxidants can maintain or decrease free radicals released by cellular metabolisms. They would avoid greater wear of sperm cells, which would endure the maintenance of speed and vigor, as observed in the results presented by Sariozkan et al. (2009)  Treatment with IGF-1 at the concentration of 100ng/mL did not present a significant difference (P>0.05) when compared with the other treatments for the analyses performed. IGF-1 is a growth factor that is associated with the increased sperm motility, as demonstrated by the results of Selvaraju et al. (2009), who evaluated the effect of the addition of 100ng/mL of IGF-1 to the semen of buffaloes, observed an increase in linear velocity and spermatic motility. Souza (2011) also obtained a positive response between the IGF-1 concentrations of the seminal plasma of gir-dairy bulls and spermatic motility, the same found by Padilha et al. (2012) when assessing the effect of the growth factor in the concentration of 100ng/mL in sheep semen. This response is partly due to the dilution of semen for the realization of cryopreservation because it promotes the dilution of the factors that compose the seminal plasma where it is contained, decreasing the concentration of this hormone. Therefore, it is expected that by recomposing its physiological values in the pre-dilution, we obtain a higher response of this variable after the freezing process.
The concentration used in the present study is per the physiological conditions pointed out by Henricks et al. (1998). However, it did not present higher spermatic motility after cryopreservation, and these results may be justified because of the existing function of this growth factor in capturing the nutrients present in the diluter, such as amino acids and sugars, which can facilitate the passage to the inner region of the cells, making the use of energy faster, leading, however, to release a greater number of free radicals, which consequently can promote cell death (Silva et al., 2013).
As for the sperm morphology of cryopreserved semen, there was no difference between treatments (P>0.05), by the Friedman test, for the major and minor defects. The use of gSH in the extender was intended to reduce the lesions that ROS can cause in sperm. One of the consequences of increased oxidative stress is the high number of spermatic defects. Thus, the addition of gSH could preserve the spermatic morphology after cryopreservation. Ansari et al. (2011), also observed that the use of antioxidants in Buffalo semen was able to maintain sperm morphology under control, as well as motility and spermatic viability. As for the addition of IGF-1 to semen, there are reports that it promotes improvement in the spermatic quality of horses (MacPherson et al., 2002), Buffaloes , and pigs . In these cases, the spermatic morphology is preserved, suggesting that IGF-1 has a crucial function in preserving the integrity of these cells, which is corroborated by the findings of Souza (2011), who found a correlation between the seminal concentrations of IGF-1 and total defects during peripuberty in gir Bulls.
The motility at time 0', 10', 20' and 30' of TTRr, in different treatments, did not differ statistically (P>0.05). These data are like those found by Peixoto et al. (2013) who, when working with semen from dogs, observed that the diluter with gSH and Trolox did not interfere in motility when compared to the control during the slow TTR test for 60 minutes. However, when evaluating the maintenance of sperm viability within the treatments comparing the initial and final TTRr times, it was observed that the control group, gSH, IGF-1, and the association had significant differences (P < 0.05) within the time, but not between treatments, decreasing motility. This answer is part of the characteristics of this variable. It was expected that the treatments with gHS and IGF-1 had less variation in the values, initial and final, of the motility, something that had not been proven.
The basic principle of antioxidants is the reduction of free radicals in the face of the challenge of increasing metabolism (Bilodeau et al., 2000). Thus, what, in theory, could have favored the maintenance of spermatic motility in the face of its addition. It would be expected greater vigor and motility at the zero moments of thawing, with the consequent decrease in motility, within the time since it would have an inverse role to the antioxidants with increased velocity, increased metabolism, consequently greater production of ROS and, finally, lower motility at the end of the evaluation. of bovine with increased diluter of gSH (0.5 and 2.0mM), observed progressive motility when compared to the control group. Bilodeau et al., (2001), used cryopreserved semen of bovine plus 0.5mM of gSH and incubated for 6 hours at 38.5°c observed the maintenance of spermatozoa motility without an external source of oxidative stress (H2O2) added to the diluter.
In the present study, it is noted that the percentage of cells with the intact plasma membrane in all treatments was low, as well as that of the undamaged acrosome, but this response may be associated with increased fluorescence of the probes used by the TRIS diluter. However, due to the low response of the sperm membranes, it should be noted that the spermatozoids with intact plasmatic and acrosomal membranes are more apt to perform fertilization (Soares et al. 2011).
Thus, it was found that gSH and IGF-1, in the concentrations used, failed to improve the quality of the acrosomal and plasmatic membrane. These results may indicate that the gSH in the 2mM concentration has no positive effect, compared to the methods used in this study. This probably occurred due to the concentration used did not supplant the decrease occurring during the seminal dilution for the cryopreservation process. According to Bilodeau et al. (2000), after cryopreservation of bovine semen, there is a reduction of 78% of the concentration of gSH and, depending on the concentration, can cause damage to bovine spermatozoa, by altering the cell osmolarity, weakening the membrane and causing rupture in this structure (Bilodeua et al. 2001). The same was noted by Stradaioli et al. (2007), who observed that in the extenders for bovine egg yolks there is a decrease in the concentration of gSH when compared with those without products of animal origin. As was found by Soares et al. (2011), noting that the different concentrations were not able to preserve membrane integrity. Sariozkan et al. (2009), when working with Holstein cattle, added to the 2mM diluter of gSH and observed the maintenance of the integrity of the spermatic membranes when compared with the control group. However, when assessing the rate of gestation in the used concentration of gSH, there was no significant difference. When referring to IGF-1, it was observed that it did not improve the maintenance of the integrity of the spermatic membranes. These data are contrary to those found by Brito et al. (2007) who observed a positive correlation between the IGF-1 concentration and the maintenance of membrane integrity. However, Selvaraju et al. (2010), when working with Buffalo semen and adding 100ng/ML of IGF-1, observed that the hormone did not affect the integrity of the acrosome despite having promoted a reduction of lipid lipoperoxidation. The same was found by Padilha et al. (2012) when evaluating IGF-1 action in sheep semen at concentrations of 100 and 250ng/mL.
It was noted that there was no difference between treatments when evaluating the percentage of cells with high mitochondrial potential. The values of this variable are important, since they are responsible for the ATP production, being indispensable to the flagellar beat for the motility of spermatozoa (Flesch and Gadella 2000).
Studies have shown that human spermatozoids have a higher rate of in vitro fertilization, and those with high mitochondrial potential have a positive correlation between motility, hyperactivation of spermatozoa, the average velocity of the route, and mitochondrial membrane potential (Kasai et al., 2002). Ahmed et al. (2016), evaluated the reproduction of buffaloes outside the reproductive period by associating the semen quality parameters with fertility after 528 inseminations and observed that the integrity of the plasmatic, acrosomal membrane and potential of the mitochondrial membrane are directly related to fertility.
As for oxidative stress, the data of the present study corroborate the reports of Selvaraju et al. (2009), where they reported that IGF-1 is responsible for being an activator of the sperm metabolism, promoting an increase in sperm motility and, consequently, increased carbohydrate metabolism. O'Flaherty et al. (1997) affirmed that the increase in the energy metabolism of sperm cells is related to the generation of a greater number of free radicals, which may affect the sperm quality and its fecundating capacity. The reactions produced naturally in the organism through the metabolism itself can generate the ROS, who's most found free radicals are: the anion superoxide (O2-), hydroxyl radical (OH-), and the metabolite peroxide of Hydrogen (H2O2) (Chan et al., 1999, Borges et al., 2011. However, Selvaraju et al. (2009) stated that when IGF-1 was added to Buffalo semen and subjected to malondialdehyde test (MDA), they observed a direct correlation with the reduction of lipid peroxidation levels.
The IGF-1 system may be involved in the transduction signal, leading to increased motility, spermatic capacitation and acrosome exocytosis (Gupta, 2005). Souza (2011) found a correlation between the concentrations of IGF-1 in the seminal plasma and the concentrations of protein peaks with affinity to heparin, which corroborates the idea of linking this hormone with the process of sperm capacitation. Thus, at the moment when the processes of sperm capacitation with alteration in sperm metabolism and motility are carried out, ROS production will also act in these processes (Soares and Guerra 2009, Borges et al., 2011, Walczak-Jedrzejowska et al., 2013. The hydrogen hydroxide and peroxide present in the sample are the main subproducts of the increase in sperm metabolism induced by the growth factor (Alves et al., 2015). Thus, when analyzing the gSH data, it was observed that 82.79% of the sperm cells did not present oxidative stress, this demonstrates that although there was no significant difference when compared with the control, the gSH was more effective than IGF-1 in maintaining the quantity of ROS. Gadea et al. (2005), when working with gSH at the concentration of 1mM and 5mM, observed that the exogenous addition of this antioxidant only takes effect when the levels of endogenous gSH are below normal levels. However, Tuncer et al. (2010) observed that when assessing oxidative stress in bovine semen plus gSH (0.5 and 2.0mM), it decreased when compared to the control group.
The pregnancy rate is following the pattern of responses presented by the other variables analyzed, which did not present the statistical difference between the treatments, framing the recommendations adopted by the Brazilian College of Animal Reproduction (Henry and Neves, 2003), which indicate the use of reeds with at least 30% spermatic motility, after thawing, so that they are classified as satisfactory. Similar results were presented by Sariozkan et al. (2009), who use the Bioxcell diluter with 2mM of gSH to freezing, maintained the seminal characteristics, however, without increasing the gestation rate (P>0.05). As well as Tuncer et al. (2010), who performed evaluations in cryopreserved semen of bovine with diluters plus gSH (0,5 and 2,0mM) observed that the pregnancy rate of 233 cows inseminated with the frozen samples did not differ between the groups (GSH with 0.5 and 2.0mM/cysteine with 5 and 10mM). In the present study, although the gSH preserved 82% of the sperm cells against oxidative stress, it was not possible to obtain a significant difference between the rates of pregnancies, because other factors may have influenced at the time of fertilization.

CONCLUSIONS
IGF-1, gSH and its association did not cause changes in spermatic motility, mitochondrial potential, plasma and acrosomal membrane integrity. However, IGF-1 increased oxidative stress. There was no increase in the pregnancy rate resulting from any treatment.  Theriogenology, v.56, p.275-286, 2001.