Impact of adding different concentrations of IGF-I and insulin to the semen extender on bull sperm quality post-cryopreservation

ABSTRACT This study aimed to evaluate the addition of different concentrations of IGF-I and insulin to egg yolk-based extender to improve bovine semen cryopreservation. Two experiments were developed to evaluate the effects of the additives in two commercial extenders, Botubov® (Experiment 1) and Triladyl® (Experiment 2), both with the same design. Three ejaculates from four bulls (n = 12) were used. Each ejaculate was divided into seven equal fractions for dilution (60x106 spermatozoa/mL) in the following treatments: CON: extender only; IGF100: IGF-I 100ng/mL; IGF200: IGF-I 200ng/mL; INS150: insulin 150µUI/mL; INS200: insulin 200µUI/mL; ASS1: IGF-I 100ng/mL + insulin 150µUI/mL; ASS2: IGF-I 200ng/mL + insulin 200µUI/mL. Semen was cryopreserved by an automated system. Post-thawed sperm were evaluated regarding motility by CASA (Computer-assisted sperm analysis), and membranes by fluorescent probes (H342, PI, FITC-PSA and JC-1). For Botubov® extender, INS150 was more efficient in preserving total and progressive motility, VCL, BCF, plasma and mitochondrial membranes. A similar response was seen when insulin was added to the Triladyl® extender, INS150 was more efficient in preserving sperm motility, plasma membrane integrity and mitochondrial potential. Thus, the addition of insulin 150µUI/mL, regardless of the composition of the extender, contributes to better preserving bovine sperm from the cryopreservation effects.


INTRODUCTION
Semen cryopreservation keeps sperm cells at cryogenic temperatures for a long term (Treulen et al., 2018) and allows the widespread use of artificial insemination in cattle (Grotter et al., 2019).However, the process of freezing and thawing the semen results in irreversible changes in sperm kinetics, membranes, and DNA integrity (Celeghini et al., 2008;Amidi et al., 2016).These changes directly reflect on the fertility of cryopreserved semen (Oliveira et al., 2014).Moreover, damage to cells during freezing may be related to osmotic stress, the formation of intracellular ice crystals, the production of reactive oxygen species or, simultaneously, the combination of all these factors (Grotter et al., 2019).
The fertilization capacity of cryopreserved sperm depends on the preservation of the integrity and functionality of the sperm structures, which are acquired by components of the extenders as ionic and non-ionic substances, responsible for maintaining osmolarity and buffering.Some components of the extenders are high molecular weight lipoproteins, present in egg yolk, crucial to avoid cold shock; intracellular cryoprotectants, such as glycerol; energy sources, represented by sugars like glucose and fructose; and other additives (Oliveira et al., 2018).
To minimize some negative effects of semen cryopreservation, several additives are studied to guarantee better preservation of sperm cells, as reported by Pinto et al. (2020) in bovine, by Dalmazzo et al. (2018) in dogs, by Banday et al. (2017) in ovine and by Berkovitz et al. (2018) in humans.However, to the best of our knowledge, no additive was shown as minimizing the cryopreservation process by turning the postthawed semen similar to in natura semen.
Bovine semen extenders supplemented with hormones such as IGF-I and insulin can improve post-thaw sperm metabolism and maximize the preservation of post-thaw sperm structures.Insulin is a protein composed of two chains (α and β, with 21 and 30 amino acids, respectively), which provides energy to sperm.This component is released into the seminal plasma, with the production of NADPH, influencing sperm capacitation and autocrine metabolism (Andó and Aquila, 2005).Autocrine metabolism is characterized by the releasing of insulin by sperm, which self-regulates glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that limits the pentose phosphate pathway (PPP), essential for sperm to acquire motility and sperm capacitation, in addition to promoting fusion between gametes (Andó and Aquila, 2005;Aquila et al., 2005).Previous studies adding insulin in the cryopreservation medium have shown better preservation of the plasma and acrosome membranes integrity, as well as the cellular metabolism (Van Tilburg et al., 2008), decreased sperm DNA fragmentation (Shokri et al., 2019) and increased sperm fertilizing capacity (Lampião and Plessis, 2008).
The insulin-like growth factor I (IGF-I) is a peptide similar to insulin and is a component of seminal plasma secreted by Leydig and Sertoli cells.IGF-I has a key role during spermatogenesis and steroidogenesis (Lee et al., 2016).Also, it preserves sperm motility, as well as plasma and acrosome membranes integrity, as demonstrated for bovine (Henricks et al., 1998), swine (Silva et al., 2011), buffalo (Selvaraju et al., 2016) and equine species with an increase in the pregnancy rate (Macpherson et al., 2002).
Although insulin and IGF-I already have beneficial effects on sperm quality during cryopreservation, the best concentration of these additives and the impact of their association have not yet to be elucidated in cryopreserved bull sperm.Based on that, this study aimed to evaluate the addition of different concentrations of insulin and IGF-I in egg yolk-based extenders (Botubov ® and Triladyl ® ) for bovine semen cryopreservation on sperm kinetics and plasma, acrosome and mitochondrial membranes, the benefit of the addition being expected even when the composition of the extenders is unknown.

MATERIAL AND METHODS
Four Nellore bulls with an average age of 55 ± 0.95 months and body weight of 921.25 ± 78.46 kg were used.The animals were kept in paddocks with pasture of Brachiaria sp. and mineral salt and water ad libitum.Concentrate was provided according to the nutritional requirements indicated for the category and age following the National Research Council (NRC, 2016).The animals were weighed every 15 days.Before the beginning of the experiment, endo and ectoparasite control was performed.This study was conducted into two experiments, according to commercial egg-yolk-based extender used: Experiment 1: Botubov ® and Experiment 2: Triladyl ® .
The experimental procedures were approved by the Ethics Committee on the Use of Animals (CEUA) of the School of Veterinary Medicine and Animal Science, the University of São Paulo-USP, Brazil (protocol CEUA number 2861160816).
In Experiment 1 (Addition of different concentrations of IGF-I and insulin to the Botubov ® extender for cryopreservation of semen), three ejaculates from each four Nellore bulls were used (n = 12).The semen was collected by electroejaculation (Autojac, Neovet ® , Uberaba/SP/Brazil), in a 15-mL graduated tube.After collection, in natura semen was kept in a water bath at 37°C during evaluation.Each ejaculate was evaluated for volume, concentration, motility, vigor, and morphology.
Semen volume was determined by the graduated tube used during collection.The sperm concentration was determined in a Neubauer chamber, under optical microscopy at 400x magnification, after the previous dilution (1:100) in a buffered formaldehyde solution (4%).Sperm motility and vigor were evaluated with a semen drop between a slide and a coverslip, under optical microscopy (Nikon, Eclipse E200) at 100x magnification.Motility was estimated as a percentage of moving sperm and sperm vigor as a score from 1 to 5, according to the recommendations for bovine (Neves et al., 2013).Sperm morphology was performed under differential interference contrast (DIC) microscopy (Nikon, Eclipse 80i), between slide and coverslip at 1000x magnification.A total of 200 cells was counted and the results were expressed as a percentage, classified into major and minor defects according to Blom (1973).
The results of the analysis of the semen in natura were used for the choice of the ejaculate for the cryopreservation and for the dilution and preparation for the cryopreservation procedure.
The choice of IGF-I and insulin concentrations used in the experimental groups was supported by the basal concentrations of these hormones in the seminal plasma, as previously described (Henricks et al., 1998;Andó and Aquila, 2005).
The semen diluted and supplemented with different hormonal concentrations were incubated for 10 minutes at 37ºC for interaction between semen and treatment.Then, semen was packaged in French straws (0.25mL) labeled with the animal number, date, and treatment.Semen cryopreservation was performed in an automatic system (TK 3000 Plus ® , Uberaba, MG, Brazil).For freezing, the semen was cooled at a rate of 0.25 C/minute until it reaches 5 C. Afterward, the straw holder was transferred to the thermal box containing liquid nitrogen until the temperature of -120 C, at a rate of -20 C/minute.Upon reaching this temperature, the straws were submerged in liquid nitrogen at -196 C. In the end, the straws were organized in racks and stored in cryogenic tanks according to each treatment and animal.
After thawing, two straws from each batch, bull and treatment were thawed in a water bath for 30 seconds at 37°C placed in microtubes, homogenized, and evaluated for motility and sperm membranes.
The characteristics of sperm motility were evaluated by Computer-Assisted Sperm Analysis -CASA (SCA ® , Sperm Class Analyzer; MICROPTIC, Barcelona, Spain).The setup was previously adjusted to evaluate bovine sperm.
A total of 200 sperm cells were counted and classified into eight categories according to the fluorescence emitted by each probe, according to Celeghini et al. (2008).After counting, the percentages of the following cell populations were considered for this study: plasma membrane integrity (PI), acrosome integrity (AI), high mitochondrial membrane potential (HMP), and spermatozoa showing plasma and acrosome membranes integrity and high mitochondrial membrane potential (PIAIHP).

In Experiment 2 (Addition of different concentrations of IGF-I and insulin to the
Triladyl ® extender for cryopreservation of semen), three ejaculates from each four Nellore bulls were also used (n = 12).Semen collection was performed by electroejaculation (Autojac, Neovet ® , Uberaba/SP/ Brazil).After collection, in natura semen was kept in a water bath at 37°C during evaluation.Each ejaculate was evaluated for semen volume, sperm concentration, motility, vigor, and morphology, as described in Experiment 1, for the screening of the ejaculate to be used and for the cryopreservation procedure.
The semen diluted and supplemented with different hormonal concentrations were incubated for 10 minutes at 37ºC and packaged in French straws (0.25 mL) labeled with the animal number, date, and treatment.The semen cryopreservation process was the same as described in Experiment 1.
Two straws from each batch, bull, and treatment were thawed in a water bath (30 sec/37 o C) placed in microtubes, homogenized, and evaluated for motility and sperm membranes.
For sperm motility, a total of 4 µL of semen were dropped between slide and coverslip (pre-heated) for reading by phase-contrast microscopy (Nikon, Model Eclipse 200) at 100x magnification.The percentage of sperm motility was estimated according to the recommendation for bovine (Neves et al., 2013) by an experienced technician.
To evaluate plasma and acrosome membranes integrity and mitochondrial membrane potential was used the same protocol described in Experiment 1.A total of 200 cells were counted and classified into eight categories according to the fluorescence emitted by each probe, according to Celeghini et al. (2008) and after counting, the percentages of the followed cell populations were considered: plasma membrane integrity (PI), acrosome integrity (AI), high mitochondrial membrane potential (HMP), and spermatozoa showing plasma and acrosome membranes integrity and high mitochondrial membrane potential (PIAIHP).
Both experiments were performed in randomized blocks.Parametric variables were tested by ANOVA and the means were compared by the Tukey test.All variables were tested for normality by Lilliefors.Variables that had subjective responses or did not show normal distribution after linear transformation were analyzed as nonparametric test, comparing the rankings using the Friedman test.The data were analyzed using the BioEstat 5.0 program.Statistical significance was defined as P ≤ 0.05.

RESULTS
The in natura sperm characteristics from each bull are presented in Table 1.In this table, are shown the mean and standard deviation from three ejaculates before semen cryopreservation.In experiment 1 using Botubov ® extender, it was found that total motility was better preserved when 150µUI/mL of insulin (BINS150) was added to the Botubov ® extender compared to the control group (BCON) and the other concentrations the IGF-I and insulin (Fig. 1).On the other hand, the addition of 200µUI/mL of insulin (BINS200) resulted in preservation of total motility similar to the control group (BCON), while all other treatments showed a decrease in total motility in relation to the BCON.The addition of 150 µUI/mL of insulin (BINS150) also preserved progressive motility better than the other additives (BIGF100, BIGF200, BINS200, BASS1and BASS2), but it was statistically similar to the BCON (Fig. 1).
In table 2, it is possible to observe that BINS150 group presented higher VCL and ALH than other groups.BCF was higher for BINS150 than for BIGF100, BIGF200 and BASS2, but it was similar to BCON.However, for VSL, VAP, LIN, STR and WOB characteristics, no effect of different concentrations of IGF-I and insulin using the Botubov ® extender was observed.BCF (Hz) 22.89±0.97ab 21.64±1.14bc 21.01±1.17c 23.60±1.14 a 22.46±0.87abc 22.85±1.17ab 22.0±1.15bc <0.0001  and acrosomal integrity (Table 3), no effect was noted of supplementation of IGF-I and insulin with the Botubov ® extender.For the sperm population with the intact plasma membrane, intact acrosome, and high mitochondrial membrane potential (PIAIHP), the BINS150 group demonstrated a positive effect on the preservation of sperm membranes when compared to the other groups.

PI
According to the results, the highest percentages of plasma membrane integrity (Fig. 4) and high Arq.Bras.Med.Vet.Zootec., v.75, n.5, p.771-786, 2023 mitochondrial membrane potential (Fig. 5) for post-cryopreservation semen were obtained when 150 µUI/mL of insulin (TINS150) was added to the Triladyl ® extender; however, these values did not differ from the TCON group.The same result was observed for the percentage of PIAIHP cells (Fig. 6).On the other hand, acrosome integrity was not affected by the addition of hormones (Table 6).

DISCUSSION
Several factors during sperm cryopreservation process affect the sperm performance after thawing, which includes the in natura sperm quality, the freeze/thaw method, and the extenders and cryoprotectants used (Grotter et al., 2019).The process of semen freezing induces physical and chemical stress, which causes damage to sperm plasma membrane, mitochondria, acrosome, motility, and morphology.All of these result in a decrease in the sperm fertilization ability (Shokri et al., 2019).In this study, IGF-I and insulin, at different concentrations, were added to two extenders egg yolk-based, to improve the preservation of function and structure of bovine sperm from cryopreservation damage, and this benefit is expected even when the definition of extenders composition is unknown.
The first experiment was carried out with a commercial extender (Botubov ® ), which has in its composition sugars, antioxidants, amino acids, egg yolk, glycerol, and water (Botupharma ® , Botucatu-SP).In the second experiment, the interaction of the additives in another commercial semen extender (Triladyl ® ) was tested; this one with sugars, TRIS (Hydroxymethyl), citric acid, buffers, antibiotics, egg yolk, glycerol, and water (Minitube ® ) in its composition.Although both extenders are based on egg yolk and have similar components, the exact composition of each one is unknown, protected by trade secrets.Thus, different interactions could occur that could show different benefits of the additives in favor of one of them.However, similar results were observed regarding the characteristics that were evaluated.The groups that received insulin at a concentration of 150 µUI/mL, in both experiments, presented better preservation of the sperm cells during freezing, reflecting in a higher percentage of sperm motility and intact cells components after thawing.
The improvement in the preservation of sperm motility with the addition of insulin, found in both experiments, can be explained by its performance, already well defined in the previous literature.Briefly, sperm motility is a process dependent on the production of ATP and the glycolytic enzymes present in the fibrous sheath of the flagella are one of the main energy suppliers.In addition, the mitochondria located in the intermediate piece are also energy suppliers by oxidative phosphorylation (Takei et al., 2014).Energy is important for motility maintenance, sperm capacitation and subsequently oocyte fertilization, as the energy is produced by the metabolism of external substrates (Ding et al., 2015).Small molecules such as monosaccharides easily cross the sperm plasma membrane bilayer, the other molecules receive support from membrane proteins that can actively or passively transport glucose (Ding et al., 2015).The presence of insulin is crucial for the glucose movement across the plasma membrane; however, when semen is diluted for cryopreservation, there is a decrease in the concentration of insulin present in the seminal plasma, so it is believed that its addition to the extender in the present study enabled the glucose to move across membranes, subsequently producing more ATP, which is essential for sperm motility.
Other studies corroborate the present, such as in humans, in which the cryopreserved semen with different concentrations of insulin (10, 100, 500 and 1000 ng/mL) increased the sperm motility percentage (Shokri et al., 2019).In other study, human semen supplemented with insulin (10 µmol) and leptin has shown a prolonged preservation of sperm motility and acrosomal reaction, thus increasing the oocyte's fertilization capacity (Lampião and Plessis, 2008).Men with insulin deficiency production had lower percentages of sperm motility and increased sperm defects, mainly from the acrosome region (Baccetti et al., 2002).The action of insulin on sperm kinetics is due to this affinity of 100 to 1,000 times for the heterologous receptors present in the sperm membranes, in this case, those of IGF-I, thus favoring sperm motility for greater glucose uptake (Clemmons et al., 1992).
In the present study, it was observed a higher percentage of cells with the intact plasma membrane, intact acrosome, and high mitochondrial membrane potential (PIAIHP) when insulin (150 µUI/mL) was added to both extenders.Such responses to the insulin action can be assured, since the plasma membrane, acrosomal and metabolism are cytological targets (Souza et al., 2012).During semen cryopreservation, between 15º to 5º C, mostly membranes alterations occur, such as rupture and loss in the phospholipid arrangements.In this interval, the plasma membrane changes its state from liquid to gel, decreasing the motility and increasing its permeability.Integral proteins, present in the plasma membrane, also suffer modulation during cryopreservation (Watson, 2000).Thus, it is believed that insulin binds to the sperm membrane receptors, which allows more protection during the cryopreservation process.
Reinforcing the results presented in this study, according to Aquila et al. (2005), the sperm mobilizes insulin from its granules at the time of Arq.Bras.Med.Vet.Zootec., v.75, n.5, p.771-786, 2023 hyperactivation, and this process activates a better source of energy to the sperm cell, which, in turn, destabilizes the components that maintain the integrity of the plasma and acrosomal membranes before acrosomal reaction.This statement was verified in the present study, as the treatments that used insulin, especially in the concentration of 150 µUI/mL, had the highest percentage of intact cells (PIAIHP).The same was observed when membranes integrity was separately evaluated for both extenders tested.Van Tilburg et al. (2008) found a significant effect of insulin on acrosome integrity.They reported that the higher the concentration of insulin was added to the freezing extender the greater is the sperm with intact acrosome, still, insulin does not induce the acrosome reaction but preserves the integrity of the acrosome, attributing the action of this by inhibiting the binding of cAMP to cAMP-dependent protein kinase and activating the phosphatidylinositol 3-, 4-bisphosphate and phosphatidyl -inositol 3,4,5triphosphate, mechanisms responsible for capacitation and acrosome reaction.However, this effect was not found in the present study.
In relation to IGF-I, no significant effect was reported of its action on the different extenders, the same was observed by Costa et al. (2020) in a study with bovine cryopreserved semen.They added 150 ng/mL of IGF-I to the extender and no effects were also observed.Differently, Henricks et al. (1998), Pan et al. (2015), Sang-Min et al. (2014), Serveraju et al. (2009), Maskarevich et al. (2014) and Padilha et al. (2012), in cattle, dogs, buffalo ram, goats and ovine, respectively, found an improvement in the sperm quality when extenders were supplemented with different concentrations of IGF-I.According to Kumar et al. (2019), in buffaloes, IGF-I increased the concentration of intracellular calcium, resulting in better progressive sperm motility.However, this was not noticed in the current study and could be explained by the difference in membranes composition, by the sensitivity to cryopreservation between species and by the concentration, IGF-I added in the cryopreservation extenders.
The addition of IGF-I and insulin combined did not improve sperm quality after thawing in both Triladyl ® and Botubov ® extenders.It was expected that the association of these hormones would guarantee better cell preservation, as IGF-I acted on sperm motility and linear speed, through the connection with receptors present in sperm, and that insulin acted on metabolism (Souza et al., 2012).The associated groups showed a lower percentage of sperm motility and a lower number of intact cells.The concentration used in these associations likely provided a cytotoxic effect to sperm, decreasing sperm motility, membrane integrity and providing greater damage to cells.

Table 1 .
Mean and standard deviation of in natura sperm characteristics considering the three ejaculates of each bull before semen dilution to cryopreservation

Table 3 .
Means (± standard error) of acrosome integrity (AI) of cryopreserved bovine semen using Botubov ® extender added with different concentrations of IGF-1, insulin, and combinations

Table 5 .
Means and standard deviation from three ejaculates of each bull of plasma membrane integrity (PI), intact acrosome (IA), high mitochondrial membrane potential (HMP) and spermatozoa showing plasma and acrosome membranes integrity and high mitochondrial membrane potential (PIAIHP) of

Table 6 .
Means (± standard error) of acrosome integrity (AI) of cryopreserved bovine semen using Triladyl ® extender added with different concentrations of insulin, and combination

Table 7 .
Means and standard deviation from three ejaculates of each bull of subjective motility (SM), plasma membrane integrity (PI), intact acrosome (IA), high mitochondrial membrane potential (HMP), and spermatozoa showing plasma and acrosome membranes integrity and high mitochondrial membrane potential (PIAIHP) of cryopreserved bovine semen using Triladyl ® extender added with different concentrations of insulin, and combinations .