Mitochondrial derived peptide humanin improved semen quality, semen freezability, antioxidant status and in-vitro fertility in crossbred bull

Abstract

The sperm quality, freezability, and fertility of crossbred bulls exhibit significant unpredictability, which consequently impacts the breeding program. The present study documents the supplementation of humanin, a mitochondria-derived peptide, on crossbred bull’s sperm quality, freezability, antioxidant status and in-vitro fertility. For this objective a total of 24 ejaculates, 8 from each of the three Vrindavani crossbred bulls, were selected based on sperm progressive motility (≥ 70%) and concentration (≥ 500 million). The selected fresh semen samples were divided into four aliquots and diluted with Tris-Fructose-Egg-Yolk-Glycerol (TFEGY) extender supplemented with various concentrations of humanin peptide; control (Group-1) without supplementation; Group II with 2.5 µM; Group III supplemented with 5 µM and Group IV supplemented with 7.5 µM. Semen quality parameters, sperm kinematics, sperm plasma membrane integrity, mitochondrial membrane potential, sperm cryo-capacitation status, ejaculate freezability rate, antioxidant status and in-vitro fertility assays were done. At the pre-freeze stage, there was no significant effect on the percentage of individual progressive motility in any group. Supplementation with 5 µM humanin resulted in an 87.5% ejaculate recovery rate after cryopreservation. At the post-thaw stage, sperm quality parameters, sperm kinematics, sperm plasma membrane integrity, mitochondrial membrane potential, sperm cryo-capacitation status, antioxidant status and in-vitro fertility were significantly improved in Group III (5 µM humanin supplementation). In conclusion, our results demonstrate that humanin supplementation in crossbred bull semen enhances semen freezability and post-thaw ejaculate recovery rate.

Introduction

Among the reproductive biotechnologies in livestock, sperm cryopreservation combined with artificial insemination (AI) are the most comprehensively used and impactful technologies. These technologies have resulted in tremendous gains in livestock productivity and animal breeding^1,2. Although sperm cryopreservation combined with AI is most commonly used, sperm cryopreservation procedures, even when using the most advanced techniques, are not always effective because they are known to be detrimental to the functionality as well as fertility of the sperm^3,4. This is because, owing to modification of osmotic equilibrium, cryo-capacitation, oxidative stress (OS), also the development of ice in the cells, a considerable proportion of gametes lose fertilizing capability after post-thaw⁵. After cryopreservation, the livability of sperm cells is diminished on average by 50 per cent, while the fertilizing capacity of sperm is also impaired by up to seven-fold³. The challenges of sperm cryopreservation are more in crossbred cattle, particularly in tropical and sub-tropical climate. In crossbred cattle bulls, poor seminal quality and freezability are key constraints^6,7, which are mostly caused by low sperm concentration, inadequate mass activity, and progressive motility of sperm. According to Loyi⁸, 70% of the semen ejaculates collected from Vrindavani crossbred bulls were of poor quality and could not be used for cryopreservation.

In Vrindavani crossbred bulls, 38%-70% semen ejaculates were of poor quality and could not be used for cryopreservation^8,9. Similarly, in half-bred (Harianan x Friesian) and three-bred cross (Harianan x Friesian x Jersey) 54% and 75% of ejaculates, respectively, were of poor freezable quality¹⁰. Furthermore, the percentage of crossbred bulls that have their ejaculate rejected due to poor semen quality ranges from 10% to 100%^11,12,13,14. Further, it was reported that sperm motility at post-freezing-thawing decreases whilst the exotic lineage of the crossbreed enhances¹⁵. On account of these factors, there is a significant disparity between supply and demand for sperm, particularly in crossbred bulls¹⁶. To improve the post-thaw quality and fertility of crossbred bull semen, several approaches have been investigated including the addition of antioxidants in extenders, early selection of good fertile bulls, freezing of good freezable ejaculates, dietary supplementation of omega-3 fatty acid etc. However, the search is on for finding an indigenous biomolecule with multifaceted properties. The major advantage of indigenous biomolecules is that they will have no or minimum negative consequences on sperm fertility¹⁷. One such biomolecule is humanin, which has recently been investigated for its cryoprotective, antioxidative and antiapoptotic role in health and reproduction^17,18,19,20.

Humanin is a 24-amino-acid peptide that is encoded by the 16 S MT- RNR2 gene of the mitochondria²¹. It was found in human male and female reproductive systems^19,22, mice²³ and rat testicles²⁴. Recently, our lab has reported humanin’s presence in buffalo and cattle bull testis and sperm^7,20. In humans, humanin was discovered to be expressed in Leydig cells, spermatocytes, spermatids, ejaculated sperm and testes^17,25. It was recently reported that supplementing a synthetic humanin, S14G-humanin (HNG), in an extender of human sperm improved post-thaw sperm livability and motility, reduced harm toward the membrane of mitochondria as well as DNA integrity, and subdued oxidative stress and caspase-3 activity in sperm after cryopreservation^17,22,26. Recently, a substantial association has been found between seminal plasma humanin levels and concentration of sperm, sperm progressive motility and quality¹⁹in humans, indicating its potential as a semen quality marker. Furthermore, in buffalo, freezable ejaculates’ seminal plasma had considerably higher humanin concentrations than non-freezable ejaculates’ seminal plasma²⁰. Lue et al.¹⁷ and Wang et al.²⁷ reported that humanin may be important in the amelioration of testicular stress and prevention of sperm injury with possible implications for male fertility preservation^26,28.

The protective function of humanin in the nervous system is well known, however, only a few studies have focused on its role in semen cryopreservation and fertility in livestock^7,20,28. Therefore, the aim of the present study was to investigate the effect of humanin supplementation to the semen of Vrindavani crossbred bulls on semen quality, freezability, antioxidant status and in-vitro fertility.

Results

Semen attributes of Vrindavani bulls at fresh stage

The average values for sperm concentration, initial progressive motility, acrosome integrity, and plasma membrane integrity at the fresh stage have been given in Table 1.

Table 1 Physico-morphological and functional attributes of Vrindavani bulls’ sperm at fresh stage (Mean ± SE; n = 24).

Effects of humanin supplementation on sperm progressive motility

Supplementing with humanin exhibited no significant (p > 0.05) effect on the sperm progressive motility at the pre-freeze stage in any group (Table 2). In all four groups, progressive motility was found to be significantly (p < 0.05) lower at the pre-freeze and post-thaw stages than at the fresh stage. At the post-thaw stage, progressive motility of sperm was found to be significantly (p < 0.05) higher in 5 µM and 7.5 µM humanin-supplemented groups.

Table 2 Effect of humanin supplementation on percent sperm progressive motility at fresh, pre-freeze and post-thaw stages of cryopreservation (Mean ± SE; n = 24).

Effects of humanin supplementation on sperm plasma membrane integrity

At the pre-freeze and post-thaw stages, significantly (p < 0.05) more HOST-positive sperm were recorded in Groups III and IV as compared to the control group (Table 3). In addition, the percentage of HOST-positive sperm was recorded to be significantly (p < 0.05) lower at the pre-freeze stage as compared to the fresh stage in control and Group II.

Table 3 Effect of humanin supplementation on sperm plasma membrane integrity at fresh, pre-freeze and post-thaw of cryopreservation (Mean ± SE; n = 24).

Effects of humanin supplementation on sperm acrosome integrity

At the pre-freeze and post-thaw stages, significantly (p < 0.05) more sperm having intact acrosome were recorded in groups supplemented with 5 µM and 7.5 µM humanin as compared to the control group (Table 4).

Table 4 Effect of humanin supplementation on sperm acrosome integrity at fresh, pre-freeze and post-thaw stages of cryopreservation (Mean ± SE; n = 24).

Effects of humanin supplementation on sperm kinematics at post-thaw stage

In comparison to the control, humanin supplementation at concentrations of 5 µM and 7.5 µM significantly (p < 0.05) improved per cent total sperm motility and percent fast sperm motility at the post-thaw stage (Table 5). The VAP and VCL were significantly (p < 0.05) higher in groups III and IV compared to the control and group II. The mean value of straight-line velocity was significantly (p < 0.05) higher in Group III as compared to the control and Group II.

Table 5 Effect of humanin supplementation on sperm kinematics at post thaw stage as assessed using computer assisted semen analyser (CASA).

Ejaculates freezability rates after humanin supplementation

The per cent recovery rate of ejaculates was 50, 54.17, 87.5, and 70.83 in Groups I, II, III, and IV, respectively, using a cut-off value of 50% post-thaw motility as per the minimal standard protocol. Humanin supplementation with 5 µM resulted in a significantly (p < 0.05) higher freezability rate (87.5%) as compared to the control and Group II (Table 6).

Table 6 Effect of humanin supplementation on ejaculates freezability rate.

Sperm mitochondrial membrane potential (MMP) at post-thaw stage

At the post-thaw stage, the percentage of sperm with high MMP was found to be significantly (p < 0.05) higher in Groups III and IV as compared to the control and Group II (Figs. 1 and 2).

Fig. 1

Effect of Humanin supplementation in extender on mitochondrial membrane potential (HMMP) of sperm at post-thaw stage (Mean ± SE). Means bearing different superscripts (a and b) differ significantly(p < 0.05).

Fig. 2

Photomicrograph of spermatozoa stained with JC-1 stain displaying red/orange fluorescence (A: High MMP) and green fluorescence (B: Low MMP) at midpiece region (1000X).

Fig. 3

Effect of Humanin supplementation in extender on capacitation status of sperm at post-thaw stage. Mean bearing different superscripts (a, b and c) differ significantly (p < 0.05).

Effect of humanin supplementation on capacitation status of frozen thawed sperm

The mean ± SE values of the “F” pattern, which indicates non-capacitated and acrosome-intact sperm, were significantly (p < 0.05) higher in Groups III and IV (Fig. 3). The mean ± SE values of capacitated sperm with intact acrosome (“B” pattern) were significantly (p < 0.05) lower in Groups III and IV than in Group II. Humanin supplementation at 5 µM led to a significantly (p < 0.05) lower percentage of sperm displaying the “AR” pattern (acrosomal reacted sperm) compared to humanin supplementation at 2.5 µM and 7.5 µM.

Fig. 4

Effect of Humanin supplementation in extender on lipid peroxidation in sperm at post-thaw stages. Means bearing different superscripts (a, b and c) differ significantly (p < 0.05).

Effect of humanin supplementation on oxidative stress status of Vrindavani bulls’ sperm at post-thaw stage

At the post-thaw stage, significantly (p < 0.05) lower sperm MDA was estimated in Groups III and IV as compared to the control and Group II (Fig. 4). Similarly, seminal plasma total antioxidant capacity was significantly (p < 0.05) higher in Groups III and IV as compared to the control and Group II (Fig. 5).

Fig. 5

Effect of Humanin supplementation in extender on total antioxidant capacity of sperm at post-thaw stages. Means bearing different superscripts (a, b and c) differ significantly (p < 0.05).

Effects of humanin supplementation on sperm’s in-vitro fertility

For the in-vitro fertility study, the control and the humanin supplementation at 5 µM and 7.5 µM were compared. Significant (p < 0.05) improvement in the zona binding ability of frozen-thawed sperm was observed in the group supplemented with 5 µM humanin as compared to the control and the 7.5 µM humanin-supplemented group (Table 7).

Table 7 Effects of humanin supplementation on in-vitro fertility (heterologous Zona binding) of frozen thawed sperm of Vrindavani crossbred bulls (Mean ± SE; n = 24).

Discussion

Cryopreservation of semen in crossbred bulls is more challenging than in purebred indigenous and exotic bulls^8,9 mainly because of cryoinjury and oxidative damage to sperm. The main site of free radical production in sperm is mitochondria, therefore, an indigenous biomolecule with mitochondria as a potential site of action is much needed to rescue the sperm from oxidative-induced damage. Humanin is a potent antioxidant, anti-apoptotic and cytoprotective indigenous biomolecule that has been detected and identified in humans, rats, mice and recently in buffalo and cattle sperm and testicular tissue. Therefore, in continuation with our previous research, the present experiment was done to rescue the Vrindavani crossbred bull’s sperm from cryo-induced damage during cryopreservation using humanin supplementation.

In the present study, the semen quality parameters (concentration, IPM, acrosome integrity and plasma membrane integrity) at the fresh stage were in concurrence with earlier reports in crossbred bulls^7,8. Sperm progressive motility, acrosome integrity and intact plasma membrane are essential for sperm fertility, induction of acrosomal reaction after fusion with the outer acrosomal membrane and fertilization of ova^29,30. Semen ejaculates for the present study were selected based on the initial IPM to avoid any experimental bias.

At the pre-freeze stage, spermprogressive motility did not differ significantly between the control and treatment groups. Katiyar et al.²⁰ reported a similar set of results, concluding that humanin supplementation in buffalo did not cause any substantial variations in progressive motility between treatment and control groups atthe pre-freeze stage. However, HOST-positive and acrosome-intact sperm were significantly higher inGroups III and IV as compared to the control and Group II. Humanin supplementation in Groups III and IV, as a powerful antioxidant, may have protected sperm from oxidative stress and resulted in higher sperm with intact plasma membrane and acrosome integrity^7,20,28. In humans, humanin supplementation in freezing media has been shown to reduce ROS and MDA generation, hence inhibiting oxidative stress reactions¹⁹. In buffalo and cattle, it has been reported that humanin-added extender led to significantly higher sperm with intact plasma membrane and acrosome integrity in comparison to the control group^7,20. Functional integrity of the sperm plasma membrane and acrosome is an important component of sperm quality and function, including their capacity to maintain homeostasis, interact with the environment, and fertilize oocytes^31,32. For a sperm to fuse with the ova, both membrane fluidity and integrity are compulsory³³. In contrast, sperm are very vulnerable to low temperature owing to the sensitivity of the plasma membrane, which is the principal source of injury in cryopreserved sperm³⁴.

At the post-thaw stage, total motility and sperm progressive motility were significantly higher in GroupsIII and IV as compared to the control and Group II.Progressive motility of sperm, when combined with other semen quality indicators, can help to predict the quality and fertility of the sperm, as it is linked to the sperm’s capacity to fertilize oocytes³⁵. However, cooling or freezing during cryopreservation disrupts plasma membranes, resulting in the loss of cations and enzymes, which reduces sperm motility and metabolic activity^36,37. Katiyar et al.²⁰ found similar results in buffalo, reporting that humanin supplementation leads to significantly higher sperm motility. Furthermore, Yang et al.¹⁷ discovered that freezing human normospermia semen ejaculates in freezing media supplemented with humanin analogue (HNG) significantly increased both progressive and total sperm motility, which is consistent with our findings. The same research also found that asthenospermic ejaculates and oligospermic patients who used HNG supplements had a substantial improvement in both progressive and total sperm motility. However, Pande et al.⁷ reported improvement in cattle sperm quality after the addition of humanin but at much higher concentrations (10 and 20 micromolar) as compared to the present study which may be because of the use of a humanin analogue and breed. Sperm motility which is strongly associated with sperm fertilizing ability, is driven by ATP produced in the inner mitochondrial membrane and transported to the microtubules to promote motility. Cryopreservation-induced decreases in post-thaw progressive motility of sperm, on the other hand, is thought to be linked to mitochondrial dysfunction³⁵. Oxidative stress because of cryopreservation damages plasma membranes of sperm, inhibits mitochondrial function, and impairs numerous axonemal proteins, resulting in loss of motility of sperm³⁸. The 4HNE and MDA are lipid peroxidation products that can diffuse into different cellular compartments and create adducted proteins^38,39. As a result of the protein adduction, the sperm cytoskeleton structure is disrupted, and the activity of the electron transport chain is impacted, resulting in sperm motility loss³⁸. These negative effects might be mitigated in this study by incorporating humanin into the semen extender, leading to an improvement in post-thaw sperm progressive motility. Humanin has been known to minimize sperm mitochondrial damage and protect sperm from ROS generation during cryopreservation^19,20.

In comparison to the non-supplemented group, humanin fortification at a 5µM concentration has improved sperm kinetic parameters such as VAP, VCL, and VSL. Findings of the present study indicated that humanin substantially improved sperm kinematic parameters which might be attributed to the intactness of the plasma membrane preserved by humanin peptide^{7,17,19,20,28}. The fertility of bull semen can be predicted using various velocity characteristics, such as VCL, VSL, and VAP⁴⁰. VCL, VSL, and VAP were correlated with pregnancy rate in frozen-thawed bull sperm. Given that it showed the strongest correlation with fertility, VAP is the most preferred sperm velocity attribute that may be used to assess sperm fertility⁴¹.

The post-thaw recovery rate of good freezable ejaculates increased significantly after humanin supplementation as compared to the control. Findings of the current study indicate that humanin fortification in the extender can improve semen freezability rate and then reduce ejaculate discard rate. Pande et al.²⁸ found that good freezable cattle semen ejaculate had a greater seminal plasma humanin concentration, which is consistent with our findings. Similarly, Rao et al.¹⁹ reported humanin levels in seminal plasma and sperm are related to sperm quality in humans. Compared to native and exotic purebreds, crossbreds have poor semen quality and freezeability^8,9 which have caused widespread semen ejaculate rejection. Forty-two (70%) of the 60 semen ejaculates from Vrindavani crossbred bulls were determined to be of poor quality and unfit for cryopreservation⁸. Furthermore, it was observed that post-thaw individual progressive motility decreased as exotic inheritance in crossbred bulls increased¹⁵. Humanin (HN), a powerful antioxidant mitochondrial-derived peptide, has recently been demonstrated to improve post-thaw semen quality in both humans¹⁹ and buffalo²⁰.

In the present study, the addition of 5 µM and 7.5 µM of humanin to semen extender resulted in a significant improvement in post-thaw HOST-positive sperm as compared to the control. Similar results were also reported by Katiyar et al.¹⁷ in buffalo and Pande et al.⁷ in cattle but at higher humanin concentration. Bull sperm have a low cholesterol level and a high unsaturated to saturated fatty acid ratio, making them vulnerable to cryo-injury⁴². In addition, cryopreservation causes disproportionate ROS production and enhances lipid peroxidation while decreasing sperm antioxidant defence activity^43,44. Lipid peroxidation produces a variety of lipid metabolites, including peroxyl radicals, alkoxyl radicals, malonaldehyde, 4-hydroxynonenal, and acrolein, which damage the plasma membrane and target neighbouring polyunsaturated fatty acids³³.In this study the improvement in plasma membrane integrity in humanin-supplemented groups (Groups III and IV) might be credited to the protective influence of humanin peptide on the structural and functional integrity of the plasma membrane at these dose rates. Humanin-supplemented groups had much lower levels of MDA than the control group, which could be the fundamental factor in humanin exerting its protective impact on post-thaw plasma membrane integrity. Furthermore, in another study, HNG supplementation to human semen freezing media led to a significantly higher post-thaw HOST-positive sperm as compared to the control in a dose-dependent manner¹⁷. In our study, at the post-thaw stage integrity of the plasma membrane did not improve in a dose-dependent mode, which is in agreement with the report of Katiyar et al.²⁰, who found that humanin supplementation did not improve post-thaw membrane integrity in buffalos in a dose-dependent style.

In order to fertilize the oocyte, sperm with an intact acrosomal membrane must undergo timely capacitation and acrosome reaction⁴⁵. Cryopreserved sperm exhibit capacitation-like alterations and are partially capacitated due to cryopreservation-induced membrane modifications⁴². The percent of post-thaw sperm with intact acrosome was significantly higher in Group III and Group IV as compared to the control group. The increased acrosomal intactness in the humanin-supplemented groups could be explained by the cytoprotective qualities of the humanin peptide. Because sperm’s plasma membrane is high in PUFA, it is more prone to free radical damage during the processes of freezing and thawing⁴⁶. In crossbred bulls, sperm cryopreservation results in decreased acrosome integrity, elevated lipid peroxidation, and excessive ROS generation⁴⁶. Humanin may maintain acrosomal integrity by lowering peroxidative damage as a powerful antioxidant^19,20.

In the present study, the proportion of sperm with high MMP was significantly higher in Groups III and IV as compared to the control and Group II at the post-thaw stage. Humanin has a role in energy metabolism, preserving mitochondrial structural and functional integrity, which could explain humanin’s protective actions in improving MMP^17,47. Katiyar et al.²⁰ revealed that a significantly higher percentage of frozen-thawed sperm containing HMMP in the humanin-supplemented groups as compared to the non-supplemented group in buffalo, which is consistent with the current study. Also, in line with the results of this study, Yang et al.¹⁷ in a study on human normospermia ejaculates, reported a significantly higher percentage of sperm with HMMP in the groups supplemented with HNG (a humanin analogue) as compared to the control. The mitochondria in the sperm midpiece region produce the majority of the ATP needed for sperm metabolism, including motility⁴⁵. Cryopreservation reduces sperm motility mostly owing to mitochondrial dysfunction^48,49. For example, lipid peroxidation reduces post-thaw sperm motility by generating adducts between lipid metabolites and mitochondrial electron transport proteins, interrupting mitochondrial electron transport and causing electron efflux, which subsequently combines with oxygen to generate more ROS in a vicious cycle⁵⁰. Lipid peroxides also disrupt the electron transport chain and harm the sperm cytoskeleton integrity during cryopreservation³⁸. Sperm motility is closely related to the percentage of sperm with active mitochondria and it is a measure of mitochondrial functionality^28,45.

In the present study, non-capacitated sperm were significantly higher in Groups III and IV as compared to the control and Group II.Since there were more non-capacitated frozen-thawed sperm in the groups supplemented with 5 µM and 7.5 µM humanin, it is likely that humanin plays a protective and beneficial role in preventing cryo-capacitation.The beneficial effects of humanin peptide in protecting sperm from freezing-thawing-induced capacitation-like changes are attributed to its potent antioxidant properties^17,19,20.The acrosome reaction and capacitation are linked to ROS formation because these free radicals control both processes³⁸. Although ROS play an important role in capacitation and acrosome reaction at appropriate levels, they are harmful to sperm function at large concentration⁵. After cryopreservation, sperm acrosome integrity impairments occurred, resulting in cryo-capacitation and premature acrosome reactivity due to abnormalities in membrane fluidity^51,52. Capacitation-like effect or ‘cryo-capacitation’ is associated with shorter lifetime along with poor survivability of frozen-thawed sperm in the female genital tract, resulting in lower fertility of frozen-thawed semen⁴². It is well known that acrosome reaction and capacitation reactions are redox regulated which are controlled by free radicals³⁸. Cryopreservation of cattle sperm, in particular, is well known to cause excessive ROS production⁴³ and decrease in antioxidant capacity⁴⁴. The oxidative stress damages the acrosome membrane after cryopreservation which leads to cryo-capacitation and premature acrosome reaction due to abnormalities in membrane fluidity^51,52. When added to the semen freezing media in humans¹⁷ and buffalo²⁰, humanin has been shown to lower ROS and MDA levels, preventing cryo-capacitation.

In this study, MDA levels were significantly lower in Groups III and IV than in Group II and the control. The current findings on LPO are in agreement with those of Katiyar (2022), who found that supplementing the extender with humanin resulted in considerably lower LPO in buffalo sperm. Also, in line with our findings, Yang et al.¹⁷ reported significantly decreased LPO levels in human sperm in treatment groups compared to the control in a study on humanin analogue (HNG) supplementation to freezing media. In addition, 5 µM humanin supplementation had a quantitatively higher TAC measure than the group treated with 7.5 µM supplementation. In line with the results of the present study, humanin supplementation resulted in higher TAC in all treatment groups compared to the control in buffalo sperm¹⁷. Similarly, Katiyar et al.²⁰ reported the highest TAC level in 5 µM humanin supplementation which is in accordance with our finding. TAC levels in bovine sperm following cryopreservation were found to be negatively associated with LPO levels⁵³. TAC and LPO were found to have an inverse relationship in bull frozen-thawed sperm⁵³. Cryopreservation causes an increase in ROS generation and a decrease in antioxidative capacity of sperm, resulting in sperm damage⁵³. Bull sperm, in particular, is protected from ROS by limited endogenous antioxidants found in seminal plasma⁴³. MDA generation in sperm is proportional to the amount of PUFAs in the cell membrane and the amount of ROS present in the semen⁵⁴. As a result, MDA is an indicator of oxidative stress and has been related to male infertility⁵⁵.

The zona binding ability of frozen-thawed sperm was significantly improved in Group III as compared to the control and Group IV. In the present study, humanin supplementation at 5 µM had significantly improved sperm functional competence and antioxidative status. This ensures that a robust pool of active sperm is preserved for zona binding and fertilization. This may have resulted in higher frozen-thawed sperm-oocyte binding efficiency in the humanin-supplemented group at 5 µM than in the control and humanin-supplemented group at 7.5 µM. Humanin also plays a role in the energy metabolism of sperm, as it preserves mitochondrial structural and functional integrity^7,17,20.

In conclusion, the present experiment has demonstrated that ‘humanin’, an indigenous biomolecule derived from mitochondria, is a potent additive to improve the quality, freezability, antioxidant status and in-vitro fertility of crossbred bull’s sperm. Humanin through its role as an antioxidant has protected the bull sperm from cryo-induced damages and increased the recovery of good freezable ejaculates. This has an implication in animal breeding improvement programmes in developing countries, which primarily rely on crossbreeding of native germplasm with exotic breeds. To fully understand the cellular and molecular mechanism of action of humanin, more investigation is necessary. Although this study provides valuable preliminary insights into crossbred bulls semen freezability, the findings are constrained by the relatively small sample size. This limitation highlights the need for future research with more extensive data collection to confirm these observations and enhance the generalizability of the findings. Also, more in-vitro and in-vivo fertility studies are needed before humanin can be added to semen extender.

Methods

Experiment location

The procedures involving animal handling, management, care and experimentation were conducted as per the norms of the Institutional Animal Ethics Committee, as established by ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly (UP), and all methods were performed in accordance with the relevant guidelines and regulations. The experiment was approved by Institute Research Committee (IRC) of ICAR-IVRI Izatnagar Bareilly vide project code-IXX15082. The experiment was conducted at the Germ Plasm Center, Animal Reproduction Division, IVRI, Bareilly (UP) in the winter season (November to February). The institute is situated approximately 564 feet above sea level. The research region undergoes alternating extremes of hot and cold weather during the summer and winter. The relative humidity in the research location ranges from 15% to 85%. The Zona binding assay was conducted at the Division of Veterinary Physiology IVRI, Izatnagar.

Animals and reagents

Three apparently healthy crossbred Vrindavani bulls (crosses of Holstein Friesian x Jersey x Brown Swiss x Hariana), 5–7 years of age kept at the Germ Plasm Center, were utilised to collect semen for this study. The bulls were kept in optimal management conditions throughout the experiment.The bulls were kept in separate pens, each measuring 12–15 square metres, with an open space. Since there were no facilities for controlling the temperature, the weather inside and outside the enclosure was the same. All the glass articles used during this study were procured from Borosil (India). IMV (France) provided the artificial vagina, latex liners, and cones. Humanin peptide (Cat No. 051588) used during this study was purchased from Biolinkk (India) and kept at -20 °C untilused. Unless otherwise noted, all chemicals used in this study were reagent grade and procured from Sigma-Aldrich (Germany).

Semen ejaculates

Ejaculates were collected twice per week and twice a day, with a 15–30 min interval between collections, using an artificial vagina, as per the routine procedure. All the study bulls were washed and cleaned before collection on a regular basis.For uniformity in sampling, ejaculates with a sperm progressive motility (IPM) of ≥ 70% and a concentration of ≥ 500 million mL^− 1 were used for the experiments (Fig. 6). A total of 24 ejaculates from three Vrindavani crossbred bulls (8 ejaculates from each) for the humanin supplementation experiment were selected based on the above-described criteria. During semen examination, the graduated semen collection tubes containing ejaculates were held at 34 °C in a water bath immediately after collection.

Fig. 6

Experiment design to investigate the supplementation of humanin in Vrindavani crossbred cattle bull semen on freezability, semen quality parameters, antioxidant status and in-vitro fertility.

Preparation of semen dilutor

Tris-Fructose-Egg Yolk-Glycerol (TFEYG; Tris(hydroxymethyl)amino-methane 3.028 g, Citric acid monohydrate 1.675 g, D-fructose 1.250 g, Penicillin G Sodium 28 mg, Streptomycin sulphate 50 mg, Glycerol 7 mL, Egg yolk 20 mL, Distilled water up to 100 mL) extender was used as semen dilutor in this study. The TFEYG extender was prepared a day before semen collection. Egg yolk, glycerol, penicillin G sodium and streptomycin sulphate were added to the extender on the day of semen collection.Before use, a magnetic stirrer was used to properly combine all the ingredients for 40 to 50 min.

Supplementation of humanin in extender

Selected semen samples were divided into four groups, Group I as the control, and Groups II, III, and IV as the treatment groups. The control group was diluted and frozen in TFEGY extender without humanin supplementation, while Groups II, III, and IV were diluted and frozen in TFEGY extender supplemented with the final 2.5 µM, 5 µM, and 7.5 µM humanin concentrations, respectively. Our laboratory’s earlier research on buffalo and Frieswal bull semen served as the basis for determining the humanin concentration for supplementation in this trial^7,20. Freshly prepared humanin-treated dilutor was kept at 34 °C in a water bath for sample dilution.

Evaluation of semen quality parameters at fresh stage

The volume of semen was determined by reading the volume on the graduated semen collection tubes. The concentration of sperm (million/mL) was determined by using the photometer (Accucell Photometer, IMV Technologies, France). The sperm concentration was used to determine the final semen dilution rate of each ejaculate to obtain a sperm concentration of 80 × 10⁶/mL in the final diluted semen. The IPM was examined at 400x magnification using a phase contrast warm stage microscope following optimal dilution of the semen with TFEYG extender so that only 15–20 sperm are present in one field. A small drop of diluted semen was put on a clean grease-free glass slide maintained at 37 °C and covered with a coverslip (18 × 18 mm) to let the semen drop spread uniformly under the coverslip. Then different fields were examined at 400X using a phase contrast microscope (Motic B-1 series). The number of progressively motile sperm was ascertained and the percentage was calculated.

Sperm acrosome integrity

The acrosome integrity of sperm at fresh stages was examined using Giemsa stain⁵⁶. An aliquot of the diluted semen sample was smeared on a clean grease-free glass slide and air-dried. The dried smear was then put into Hancock’s fixative (sodium chloride 10 g, sodium bicarbonate 0.5 g, formalin 125 mL, distilled water up to 1000 mL) in a slide staining jar for 15 min. The smear was then washed in slow-flowing tap water for 15 min, rinsed with double-distilled water, and air-dried in a vertical position. The dried smear was kept in a slide staining jar containing working Giemsa stain solution (Giemsa Stock solution 3.0 mL, Sorenson’s 0.1 M phosphate buffer 2.0 mL anddouble glass distilled water 35.0 mL) for 3 to 4 h. Smears were removed from the Giemsa stain solution and washed in slow-running tap water for 15 min, then rinsed quickly in distilled water and immediately air-dried in a vertical position. Giemsa-stained slides were then examined by bright field microscopy under oil immersion with a100X objective. For each sample, two hundred sperm were analyzed and categorised as either intact or damaged acrosomes. The acrosome was regarded as intact if the Giemsa stain was equally dispersed over the sperm anterior up to the equatorial segment, while the acrosome with swelling, knobbing, denudation, and ruffling was considered damaged. Percentage of acrosomal intactness was calculated for each sample.

Sperm kinematics using computer-assisted sperm motion analysis (CASA)

The Computer Assisted Semen Analyzer (CASA) (Minitube, Tifenberg, Germany) with AndroVision^® software Version 6.1 (Minitube, Tifenberg, Germany) was used to measure the kinetic and motility parameters of the sperm. The CASA has a ZeissAxioscope Microscope (Germany) with a 20x negative phase contrast and blue filter for measuring sperm motility and kinetics. In order to maintain a constant temperature of 37 °C, a thermostatic stage was installed in the microscope. The sperm motility and kinetic characteristics were recorded in at least 10 fields with a minimum of 2000 sperm after the chambers were loaded. The parameters recoded were total motility, progressive motility, progressive fast motility, progressive slow motility, progressive circular motility, local circular motility, immotile sperm, the average path velocity (µm/s; VAP), straight line velocity (µm; VSL), curve linear velocity (µm/s; VCL), amplitude of lateral head displacement (µm; ALH), beat cross frequency (Hz; BCF), Wobble (WOB), straightness (STR), and linearity (LIN). The setting of the software was in accordance with the standard recommendations.

Semen extension and equilibration

The TFEGY extender was used to dilute the ejaculates, resulting in an extended semen that contained 80 × 10⁶ sperm per millilitre for each treatment group. Final diluted semen in all treatment groups was filled into 0.25 mL French mini straws with the help of a semi-automatic filling machine. After that, the straws were sealed using poly vinyl alcohol powder (IMV, France). The semen-filled straws were then dipped into water warmed at 35 °C in a plastic bread box with 3/4 of it filled with water. For equilibration and glycerolization, the straws were placed in a cold-handling cabinet at 4 °C for 4 h for equilibration.

Semen evaluation at pre-freeze stage

At the pre-freeze stage, semen ejaculates in all treatment groups (control and humanin supplemented) were evaluated for IPM and acrosomal integrity using methods described earlier. In addition, the hypoosmotic swelling test (HOST) was performed at the pre-freeze stage to assess sperm plasma membrane integrity.

Hypo-osmotic swelling test (HOST)

Sperm plasma membrane integrity was determined by using the hypo-osmotic swelling test⁵⁷. The of hypoosmotic solution osmolality was adjusted to 150mOsm/L. The hypo-osmotic solution having 150 mOsM was prepared from 13.51 g fructose and 7.35 g trisodium citrate per litre of double distilled water and stored at 4 °C until use. At the time of analysis, 1.0 mL of HOST test solution was poured into a glass sugar tube to which 100 µL of diluted semen was added and mixed well. The sperm suspension was incubated at 37 °C for 1 h in a water bath (Minitube, Germany). After incubation, a drop of eosin-Y stain was added and mixed well with the sperm suspension. Then a small drop of sperm suspension was taken from the bottom of the tube and smeared on the warm, clean, grease-free glass slide and air-dried. The cover slip was applied over the dried smear. Two hundred sperm cells for each sample were assessed under high-power magnification (400X) of a light microscope and different tail swelling and curling patterns were recorded. The same procedure was used for semen samples at both pre-freeze and post-thaw stages for each treatment group. The per cent HOST response of the sperm was determined according to the presence of distinct categories of swelling in the tail of the sperm (Prasad et al., 1999) as detailed below.

Pattern A: No swelling, complete loss of membrane function. Pattern B: Swelling at the very tip of the tail. Pattern C: Different types of hairpin swelling. Pattern D: Complete swelling of the tail. The sperm having B, C and D patterns of tail swelling were considered HOST positive while those displaying pattern A were considered as HOST negative. Then the percentage of HOST-positive sperm was calculated as:

$$\% {\text{HOSTpositive}}\,=\,\frac{{{\text{Number}}\;{\text{of}}\;{\text{swollen}}\;{\text{sperm}}}}{{{\text{Total}}\;{\text{sperm}}\;{\text{counted}}}} \times 100$$

Semen cryopreservation

After equilibration, the straws were frozen for 7 min in an automatic programmable freezer (Mini Digitcool, IMV Technologies) until the temperature reached − 140 °C and then plunged into liquid nitrogen (-196 °C) and stored till further post-thaw evaluation.

Semen thawing and post-thaw evaluation

Thawing of the straws was done in water at 37 °C for 30 s in a bread box and the semen content was then poured into a sugar glass tube held at 37 °C in a dry bath. Semen quality parameters including post-thaw sperm motility, acrosome integrity (Giemsa stain) and functional integrity of sperm plasma membrane (HOST) were evaluated according to the methods described previously. Evaluation of mitochondrial membrane potential and cryo-capacitation status of sperm were also conducted at the post-thaw stage. In addition, assessment of the oxidative stress status of the sperm and seminal plasma were performed at this stage.

Mitochondrial membrane potential (MMP)

The mitochondrial membrane potential of sperm at the post-thaw stage was evaluated using the fluorochrome 5,5’,6,6’-tetrachloro-1,1’,3,3’- tetraethylbenzimidazolylcarbocyanine iodide (JC-1) following the method described by Katiyar et al.²⁰ with minor modifications. To make the stock solution, 1 mg of JC-1 was dissolved in 1 mL of DMSO and kept at -20 °C. Just before examination of samples for MMP, the working solution of JC-1 was made by mixing 90µL of phosphate buffered saline solution (PBS) into 10µL of JC-1 stock solution. After thawing of the semen, 10 × 10⁶ sperm (12.5 µL of frozen-thawed semen) were washed twice with HTF-HEPES buffer solution using centrifugation at 1000 g for 5 min at 37 °C. The sperm pellet was then resuspended and thoroughly mixed with 200µL HTF-HEPES solution. Briefly, a 10µL aliquot of the resuspended semen sample was thoroughly mixed with 20µL of JC-1 working solution and then incubated at 37 °C for 15 min in a dry bath. The sperm suspension was placed on a clean, grease-free glass slide and a smear was prepared. After adding a drop of anti-fade agent (DABCO) the cover slip was placed over the slide. Sperm evaluation was performed using a fluorescent microscope (MT6300, Meiji Techno, Japan) under 400x power magnification. For each sample, two hundred sperm cells were evaluated and divided into two groups: those with high MMP emitting bright orange fluorescence through the sperm midpiece (green filter), and those with low mitochondrial membrane potential emitting green fluorescence through the sperm midpiece (blue filter). Both images from one field were combined to create an image showing sperm with high and low MMP. The percentage of sperm with high MMP was calculated.

Capacitation status

The capacitation status of the frozen-thawed sperm was evaluated using chlortetracycline (CTC) fluorescent staining as per the procedure described by Katiyar et al.²⁰. Briefly, 10µL of sperm suspension was mixed by pipetting with the same volume of CTC solution in a centrifuge tube. Then the mixture was incubated at 37 °C for 5 s in a dry bath. The CTC solution was freshly prepared daily and stored at 4 °C until used and brought to room temperature before being added to the sample. After incubation, an equal volume of the 12.5% glutaraldehyde was added into the sperm suspension. Finally, 10 µL of the sperm suspension was placed on a clean, grease-free glass slide and a thin smear was prepared. A drop of antifade agent (DABCO) was added to the smear in order to prevent the declining of CTC fluorescence and cover slip was applied over the smear. The sperm were observed under 1000x magnification power using a fluorescent microscope (MT6300, Meiji Techno) equipped with PROGRES GRYPHAX software (JENOPIK, Germany). At least 200 sperm per semen sample were counted and classified into three CTC staining patterns. Pattern F: sperm with uniformly green fluorescence over the whole head (non-capacitated sperm). Pattern B: sperm with a green head fluoresces, but a fluorescence-free band in the post-acrosomal region (capacitated sperm). Pattern AR: sperm with a dull green fluorescence signal over the entire head, except for a thin band of fluorescence along the equatorial segment (acrosome-reacted sperm).

Seminal plasma collection

To determine the oxidative status of frozen-thawed semen samples, 1 mL of thawed semen from each experimental group was put into a 1.5 mL microcentrifuge tube and centrifuged at 4000 g for 20 min at 5 °C. The supernatant and pellet were separated and kept in sterilized tubes at − 20 °C until used for estimation of lipid peroxidation and total antioxidant capacity, respectively.

Malonaldehyde (MDA) assay

The lipid peroxidation of sperm at post-thaw stage was determined by measuring the formation of malonaldehyde (MDA) using the thiobarbituric acid-trichloroacetic acid (TBATCA) assay according to the procedure described by Katiyar et al.²⁰. The sperm pellet was resuspended in a variable volume of phosphate buffer saline (PBS, pH adjusted to 7.2) in order to achieve a 20 million/mL sperm concentration in the suspension. Then 1 mL of the sperm suspension was added to 2 mL of the TBA-TCA reagent (trichloroacetic acid 37.5 mL, thiobarbituric acid 0.375 g, 1NHCL 25 mL, distilled water up to 100 mL) in a glass test tube and mixed thoroughly. This was followed by boiling the mixture for an hour in boiling water. After cooling the mixture, which turned yellow in colour due to boiling, 1 mL of the suspension was poured into a centrifuge tube and centrifuged at 300 rpm for 10 min. Finally, the supernatant was separated and used for measurement of absorbance at 535 nm using a spectrometer (Eppendorf BioSpectrometer kinetic). The TBA-TCA reagent was considered as blank. The following formula was applied to calculate the concentration of MDA in the sperm pellet.

$${\text{MDA}}(\mu {\text{mol}}/{\text{mL}})=\frac{{{\text{OD}} \times {{10}^6} \times {\text{total}}\;{\text{volume}}\;{\text{of}}\;{\text{reaction}}\;{\text{mixture}}\;(3\;{\text{mL}})}}{{1.56 \times {{10}^5} \times {\text{total}}\;{\text{sample}}\;{\text{volume}}\;{\text{(1}}\;{\text{mL}})}}$$

Where 1.56 × 10⁶ µmol/cm³ is specific absorbance coefficient.

Total antioxidant capacity (TAC) assay

The ferric reducing/ antioxidant power (FRAP) assay was used to determine seminal plasma total antioxidant capacity⁵⁸. The principle of the Ferric reducing/ antioxidant power (FRAP) assay is based upon the formation of the ferrous form through the reduction of the ferric tripyridyl triazine complex at low pH resulting in an intense blue colour of the solution. Initially, 3 mL of the working solution of FRAP reagent [(A. Acetate buffer 300 mM pH 3.6 (Sodium Acetate Trihydrate 0.31 g, Acetic Acid 1.6 mL and distilled water up to 100 mL); (B. 2, 4, 6-tripyridyl-s- triazine312 mg in 100 mL 40 mM HCL); (C,541 mg FeCL3.6H2O in 100 mL distilled water) was prepared by mixing a, b and c in the ratio of 10:1:1 at the time of use] and 100 µl of the seminal plasma were mixed in a glass test tube and vortexed. Then absorbance was measured at 593 nm using a spectrometer (EppendorfBioSpectrometerkinetic) at 0 min. The samples were put in a dry bath at 37 °C for four minutes and absorbance was measured again. The standard was ascorbic acid (1 M) processed similarly to the seminal plasma sample. Three mL of the working solution of FRAP was taken as a blank.

$$\begin{aligned} {\text{Sample FRAP value}}\;{\text{(}}\mu {\text{mol/mL)}} & \;{\text{ = }}\;{\text{(Change in absorbance of sample from 0 to 4 minute/}} \\ & \;\;{\text{Change in absorbance of standard from }}0\;{\text{to 4 minute)}} \\ & \;\;\; \times {\text{ FRAP value of standard}}\;{\text{(1M)}} \\ \end{aligned}$$

The FRAP value of 1 M ascorbic acid (standard) solution is 2000.

Sperm zona binding assay

Frozen-thawed sperm of the control and best treatment groups were subjected to a heterologous zona binding assay using buffalo oocytes⁵⁹. Ovaries were collected from buffaloes slaughtered at local abattoirs. Ovaries were transported to the laboratory in sterile isotonic normal saline solution at 30–35 °C supplemented with penicillin G and streptomycin. Collected ovaries were then washed in warm, clean, normal saline solution supplemented with antibiotics. Using a 10 mL syringe containing oocyte collecting medium (OCM, NaCl 136.89 mM, KCl 2.68 mM, KH₂PO₄ 1.46 mM, NaH₂PO₄ 8.09 mM, CaCl₂.2H₂O 0.90 mM, MgCl₂ (Anhydrous) 1.00 mM, D-glucose 5.54 mM, Sodium pyruvate 0.32 mM, Gentamicin (50 µg/ml) 500 µl Phenol red 10 µg/ml) and an 18-gauge needle, follicular fluid was aspirated from surface follicles measuring > 2 mm in diameter on buffalo ovaries. The cumulus oocyte complexes (COC) were then placed in a 50 mL centrifuge tube and kept in a CO₂ incubator at 39 °C, 5% CO₂, and 95% humidity for 30 min. The COC-containing sediments were put into square searching petri dishes containing OCM after the supernatant was decanted. Oocytes characterized by compact, multi-layered COC and dark homogeneous cytoplasm were selected in a petri dish with OCM using a stereozoom microscope. The selected oocytes were washed up to seven times in OCM medium then washed in oocyte maturation media up to 5 times. For oocyte maturation, around 10–12 oocytes per drop were transferred to petri dishes containing a drop of maturation media covered with mineral oil and cultured at 39 °C, 5% CO₂ and 95% humidity for 24 h. After 24 h, ova were examined for maturation using a stereo microscope on the basis of their cumulus cell expansion (Liu et al., 1991).

Sperm capacitation and in vitro insemination of matured oocytes

Frozen-thawed sperm in a modified TALP as a capacitation medium were incubated at 37 °C in 5% CO₂ and 95% humidity in an Eppendorf tube to induce capacitation. To remove dead cells, sperm were washed in non-capacitating medium (NCM, KCl 0.201 g, KH₂PO₄ 0.04 g, Na₂HPO₄ 1.150 g, NaCl 8.00 g, Glucose 0.999 g, Pyruvate 0.11 g in 100 mL solution). The sperm were then washed twice with TALP by centrifugation at 170 g for 10 min and supernatant was removed. The resulting packed sperm were then resuspended in fertilization TALP (NaCl 114 mM, KCl 3.20 mM, NaH₂PO₄ 0.34 mM, CaCl₂.2H₂O 2.00 mM, MgCl₂ (Anhydrous) 0.50 mM, NaHCO₃ 25.00 mM, Sodium lactate 1.86 µl/Ml, Gentamicin 50 µg/mL, Phenol red 10 µg/mL) and the concentration of sperm was adjusted to 5 × 10⁷ sperm per mL. The sperm suspension was then incubated for up to six hours in an Eppendorf tube at 37 °C, 5% CO₂, and 95% humidity under mineral oil. Matured oocytes were pipetted using a capillary tube into a disc with fertilization TALP and washed up to 6 to 8 times. About 10–12 washed oocytes were placed into 50 µl of fertilization TALP media, to which 20–30 µl of the capacitated sperm suspension was added. Then oocytes and sperm were co-incubated for 18 h at 39 °C in a 5% CO₂ air atmosphere and 95% humidity incubator. About 6–7 h later, washing was done two to three times by replacing the fertilization TALP of the droplet to remove free and loosely bound sperm to the zona pellucida. Droplets containing oocytes and sperm were then returned to the CO₂ incubator for the remainder of the incubation time.

Evaluation of sperm zona binding

The attached sperm were removed two hours after sperm addition, and new BO medium was introduced to the droplets to remove sperm that were loosely bound with zona pellucidae. After co-incubation, oocytes were washed once and put on the clean glass slide. The zona pellucida and any sperm bound to it were fixed for 10 min using 2.5% glutaraldehyde in PBS and washed in PBS. The slide was stained with Hoechst fluorescent dye 33,342 (10 µg/mL) for 10 min, washed in PBS and covered with a coverslip. The number of sperm bound to an oocyte was determined under an epifluorescence BH-2 microscope equipped with DMU filters (x 200) and the percentage of bound sperm was determined for each group of frozen-thawed semen.

Statistical analysis

The data were analyzed for normal distribution by the Shapiro-Wilk test. Homogeneity of variance of data was analyzed by employing the Levene’s test. The means of the groups were analyzed by one-way repeated measure ANOVA using IBM Statistical Package for the Social Sciences (SPSS) v27 (SPSS Inc.; Chicago, Illinois, USA). Results are presented as mean ± SEM. Pearson’s Chi-square test of independence was applied to see the relation between freezability and humanin supplementation. The differences were considered significant at p < 0.05. Statistical analysis was performed using IBM Statistical Package for the Social Sciences (SPSS) v27.