Tris and Trehalose based modified cryopreservation dilutor in block freezing improves cryo-survivability and functionality of Sahiwal bull semen

doi:10.21203/rs.3.rs-5209426/v1

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Tris and Trehalose based modified cryopreservation dilutor in block freezing improves cryo-survivability and functionality of Sahiwal bull semen

https://doi.org/10.21203/rs.3.rs-5209426/v1

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Our study aimed to enhance cryoprotection and improve post-thaw sperm quality by using a new freezing protocolthat combines trehalose and glycerol.The semen ejaculate from each bull was collected and split into three aliquots.Split one was the control group (C), in which semen was extended in a tris fructose egg yolk glycerol (TFEG) extender and underwent the conventional freezing protocol.In split two (T1) and split three (T2) groups, the semen was diluted in tris fructose egg yolk extender containing 25% trehalose, with glycerol 5% in both groups. Split two (T1) underwent conventional freezing, whereas split three (T2) underwent aluminium block freezing.After freezing-thawing, the progressive sperm motility,viability, acrosome integrity, sperm velocity and path parameters were significantly (p ≤ 0.05) improved in T2 among groups. Additionally, the degree of oxidative stress was substantially lower in T2 among groups.The present study's findings revealed the promising role of trehalose (25%) and glycerol (5%) in tris-based extender in achieving aluminium block freezing, as it significantly improved the post-thaw sperm survivability, and this method can suitably be used for quality frozen semen production.

Bull

Cryoinjuries

Glycerol

Kinematics

Sperm

Trehalose

Cryopreservation of spermatozoa is an age-old novel process of preserving male germplasm. It is a significant way of disseminatingsuperiorgermplasm for faster species propagation.Despitemuch development in cryopreservation techniques and their process, cryoinjuries are still evident during the cryopreservation of sperm. They are the primary cause behind the reduction in the functional competence of spermatozoa and, consequently, the reduction in conception through the use of frozen semen. The irreversible changes occurring during freezing-thawing are membrane alteration, acrosome, mitochondrial damage, cryocapacitation,and apoptotic-like changes, eventually resulting in diminished sperm fertilizing competence (Peris-Frau et al. 2020).

Several strategies have been planned and executed for different species of animals to address the issue of cryoinjuries in sperm.Techniques such as vitrification in stallions with a mixture of cryoprotectants have been tried (Diaz-Jimenez et al. 2018; Hidalgo et al. 2018; Consuegra et al. 2019). Additionally, in humans, sperm preservation advancements have been observed through methods like vitrification without cryoprotectants (Isachenko et al. 2011), leading to improvements to a certain extent. The dilution, freezing, and thawing techniques are essential to the success of semen cryopreservation protocol. The rate at which the semen cools is crucial to the freezing process. Moreover, exposure duration and straw height from liquid nitrogen may significantly affect the cooling rate (Du et al. 2021).Rapid freezing protocols have resulted in higher post-thaw semen quality, including increased vitality, motility, and DNA integrity, than traditional slow freezing (Liu et al. 2022; Ba-Awadh et al. 2023).

Additionally, studies comparing ultra-rapid frozen sperm with conventional slow-frozen sperm have shown that while fast freezing methods may reduce sperm motility, the addition of cryoprotectants like disaccharides can moderately protect sperm quality during rapid freezing processes (Perez et al. 2022). Overall, the evidence suggests that fast-freezing techniques can offer improved post-thaw semen quality and are advantageous for sperm cryopreservation compared to traditional slow-freezing methods. Hence, in the present study, fast freezing was done to reduce equilibration time, vapour freezing time, and the distance of straws from liquid nitrogen (LN₂). Apart from this, aluminium blocks were used to direct the contact of strawsto capture the latent heat of energy released during freezing effectively. Freezing is an exothermic process that releases latent heat of fusion and starts from the periphery to the center. This latent heat goes to the ice front and causes a repeat freeze-thaw melt cycle, which harms the cell (Saragusty 2015). Aluminium is a heat-conductive metal that captures this latent heat quickly, thereby avoiding cell damage as reported by Arav and Saragusty (2016). As in the present study, the semen samples were exposed to fast freezing, and a combination of cryoprotectants (trehalose and glycerol) was used in the tris fructose egg yolk extender to combat the freezing stress during fast freezing. Trehalose was used because it has been used in extenders in various studies for achieving fast freezing (Diaz-Jimenez et al. 2018; Hidalgo et al. 2018; Consuegra et al. 2019).Trehalose is a disaccharide of glucose found in high concentrations in many organisms capable of surviving complete dehydration (Iturriaga et al. 2009). Various studies demonstrate that trehalose improves the functional parameters of sperm after fast freezing, as it helps in maintaining a higher percentage of total and progressive motile sperms as compared to traditional freezing methods (Hu et al. 2009; Schulz et al. 2017; Gholami et al. 2023). Trehalose better protects membrane functionality than glycerol and sucrose (Starciuc et al. 2020). Trehalose has been shown to enhance fluidity and induce reorganization of the sperm membrane through its interaction with phospholipids in the plasma membrane during cryopreservation (Bakas et al. 1991).

As far as glycerol is concerned, in addition to depolymerizing actin in the sperm cytoskeleton, glycerol has been observed to be cytotoxic above a specific quantity (Garcia et al. 2012). It was seen that5% glycerol (with 1% fructose) increased the quality of frozen-thawed sperm and the fertility of buffalo (Almeida et al. 2017), Nellore bulls (Almeida et al. 2017), Crossbredbull (Batra et al. 1981) and Mithun bull (Bosfrontalis) (Baruah et al. 2013).

In semen freezing,the role of both permeable and non-permeable cryoprotectants is growing as they impart less toxicity to sperm (Isachenko et al. 2003). A study reported the use of trehalose and glycerol as cryoprotectants and modified the freezing process for poor, freezable Sahiwal semen with encouraging results (Sethi et al. 2024). In addition another study reported that fast freezing in bull semen beats traditional freezing to preserve sperm function characteristics (Baiee et al. 2020). The purpose of the current study was to evaluate the impact of block freezing on the cryopreservation of bull semen when combined with non-permeable (Trehalose) and permeability (Glycerol) cryoprotectants.

Chemicals and reagents

Tris, glycerol, benzylpenicillin, citric acid, EDTA, fructose, KCl, NaCl, MgCl₂, NaH₂PO₄, and streptomycin were procured from Sisco Research Laboratories (SRL, Mumbai, India), ethanol (Merck). Trehalose was purchased from SRL, Mumbai, India (Product Code: 91094, CAS No. 6138-23-4); fluorescent stains were procured from Sigma-Aldrich® (Milano, Italy).

Experimental bulls and semen collection

The study was conducted at the Artificial Breeding Research Centre (ABRC), ICAR-National Dairy Research Institute, Karnal (Haryana), India. A total of forty-eight ejaculates (N = 48) were collected from four healthy Sahiwal bulls twice a week using an artificial vagina under standard semen collection procedures. The bulls were managed under standard feeding practices with a regular vaccination schedule to control scheduled diseases.

Immediately after collection, the ejaculates were transferred to the water bath at 32–34˚C for further processing. The collected ejaculates were screened and, after qualifying the optimal standards (progressive motile spermatozoa > 70%,viability > 85%, and sperm concentration > 800 million/mL), further processed for freezing-thawing. Semen dilution and addition of trehalose

We carried out a pilot study to determine the trehalose concentration and block freezing methodology. The pilot study findings are provided in the supplemental file. During the pilot study, two extenders were made independently: Extender 1 (E1) with trehalose, citric acid, 1% fructose, 20% egg yolk, and 5% glycerol (280 mOsm/l) and Extender 2 (E2) with tris, citric acid, 1% fructose, 20% egg yolk, and 5% glycerol. Different combinations of these solutions were prepared by substituting extender 2 with extender 1 (both extenders were prepared with the same molarity to ensure a consistent final solution molarity of 280 mOsm/l). Four extenders containing 25% (G1), 50% (G2), 75% (G3), and 100% (G4) trehalose were prepared by mixing E1 and E2 extenders in the ratio 1:3, 1:2, 3:1, respectively. In all four extender combinations, glycerol was maintained at 5%,and block freezing with an aluminium block was done in all the samples.

The pilot research consisted of three parts (described in Fig. 1). In part one (Fig. 1a), standardization of trehalose buffer with different combination of freezing time (6, 8 and 10 minutes) and distance between liquid nitrogen (LN₂) and aluminium block (1, 2, 3 and 4 cm) was done while the equilibration period was kept constant i.e. 4 hour at 4˚C. After dilution, semen samples from all the four groups G1, G2, G3 and G4 were packaged in 0.25 mL TBS straws using an MRS I filling sealing machine (IVM, L'Aigle, France). Straws from all four groups (G1, G2, G3, and G4) were then placed within holes drilled in the aluminium block, as shown in Fig. 2 and stored at 4°C for equilibration for 4 hours. Thereafter the blocks containing straws of different groups were subjected to block freezing at varying time (6 minutes, 8minutes and 10 minutes) and at varying distance from LN₂ (1 cm, 2cm, 3cm and 4cm). This part demonstrated that semen extended in extender containing 25% trehalose with block freezing done for 6 minutes at a distance of 2cm from LN₂ preserved the sperm motility better (detailed results shown in supplementary files).

In the second part of the pilot study (Fig. 1b), standardization of equilibration period with freezing time (6 minutes) and 2 cm distance between block and LN₂ was done. For this, semen was extended in G1 extender containing 25% trehalose and was filled in straws. The straws were then kept inside holes made in aluminium blocks and were subjected to a varying equilibration period (1, 2, 3 and 4 hours) at 4°C. After this, the block containing straws was subjected to block freezing for 6 minutes at 2cm distance from LN₂. This part demonstrated that a 2 hours equilibration period preserved the sperm motility better than the rest.

Figure 1a. Pilot study. Standardization of Trehalose buffer with different combination of freezing time (6, 8 and 10 minutes) and distance between LN₂ and aluminium block (1, 2, 3 and 4 cm) while equilibration period is constant i.e. 4 hour at 4°C (N = 20)

In part third of pilot study (Fig. 1c), ejaculate was split into two. In first split group, semen was extended in E2 extender (tris, citric acid, 1% fructose, 20% egg yolk, and 5% glycerol ) containing no trehalose and in second split group, semen was extended in G1 extender containing 25% trehalose (E1:E2 = 1:3). Both the split groups were subjected to conventional and block freezing. It was seen that sperm post-thaw motility was significantly higher in the following groups: E2 extender with conventional freezing, G1 containing 25% trehalose with conventional freezing and G1 containing 25% trehalose with block freezing. Hence, these groups were finally used in the main experiment as control, treatment 1, and treatment 2, respectively. The pilot research data has been given in a supplementary file.

For the main experiment, after the initial microscopic assessment of mass motility and initial progressive motility, a photometer (IVM, L'Aigle, France) was used to evaluate the sperm concentration of all the samples. From each ejaculate, three splits were made, i.e., split one (Control; C), split two (treatment 1; T1), and split three (treatment 2; T2). The split semen sample in control was extended inTris fructose egg yolk glycerol extender (TFEG: 280 mMtris, 20% egg yolk (v/v), 1% fructose (w/v), and 6.4% glycerol v/v) extender with the conventional freezing protocol to 80 million sperm/ml to be frozen in 0.25ml in IMV TBI mini straws. The split semen samples in T1 and T2 were extended in an extender containing 25% trehalose and 5% glycerol. To prepare this extender, two extenders E1 with trehalose, citric acid, 1% fructose, 20% egg yolk and 5% glycerol (280 mOsm/l) and E2 with tris, citric acid, 1% fructose, 20% egg yolk and 5% glycerol (280 mOsm/l)were prepared. The final extender was made by mixing 75% E2 extender and 25% E1 extender. The control (C) and T1 group semen straws were subjected to conventional freezing (Equilibration at 4˚C for 4 hours; a distance of straws from LN₂ vapours (4cm) and time of exposure of straws to LN₂ vapours (10minutes). However, T2 semen straws were first put inside the holes made in aluminium blocks and were then exposed to block freezing protocol (Equilibration at 4˚C for 2 hours; a distance of blocks containing straws from LN₂ vapours (2cm) and time of exposure of blocks containing straws to LN₂ vapours (6 minutes). A diagrammatical presentation of the main experiment has been illustrated in Figs. 2 and 3.

Semen equilibration and freezing

The equilibration and freezing protocol differed for control and treatment samples. The samples were subjected to filling and sealing in French mini TBSstraws (0.25 ml, IVM, France) at 2–4˚C. The control and T1 split samples were then equilibrated at 4°C for 4 hours; after equilibration, direct vapour freezing for 10 minutes was done in a straw distribution rack. The distance between the level of LN₂ and the rack containing straws was 4 cm. The straws were immersed in liquid nitrogen (-196ºC) and stored until thawing for evaluation.

The T2 split sample straws were put inside the holes made of aluminium blocks, equilibrated at 4ºC for 2 hours, and finally underwent block freezing. The block was kept for vapor freezing in a wide-mouth LN₂ container, keeping a distance between the aluminum block and LN₂ to 2cm. Block Vapour freezing was done for 6 minutes, after which the straws were removed from the aluminium blocks and immediately dipped into LN₂.

Post-thaw evaluation of the semen

Frozen semen straws were thawed at 37°C for 30 seconds (CITO warm water thaw unit) following twenty-four hours of cryopreservation as reported by Harrison and Vickers (1990). Thawed semen samples were analysed for progressive motility, viability, membrane integrity, acrosome reaction, lipid peroxidation, and sperm kinematics.

Assessment of sperm progressive motility

Progressive motility of individual sperm was assessed at the pre-freeze and post-thaw stages. A phase-contrast microscope with a power objective (40X) was used to analyse a drop of extended semen placed on warmed glass slides. Sperm were observed in at least five fields on a thermostatic stage at 37^°C, and progressive motility was expressed with 5% accuracy. At post thaw stage sperm progressive motility was analyzed at 0, 30, 60, 90 and 120 minutes (post thaw incubation test).

Assessment of membrane integrity

Sperm viability was assessed by CarboxyfluoresceinDiacetate-Propidium Iodide (CFDA-PI) fluorescence staining (Pawshe et al. 2017), while HOS (Hypo-osmotic swelling) response (Jeyendran et al. 1984) evaluates biochemical plasmalemma activity as an intact plasmalemma.

Assessment of sperm acrosomal integrity

The acrosome integrity of spermatozoa was assessed using the fluorescent FITC-PSA staining method (Swain et al. 2017).

Assessment of sperm lipid peroxidation by malondialdehyde (MDA) estimation

The spermatic lipid peroxidation was measured by malondialdehyde (MDA) concentration using the procedures of Buege and Aust (1978) and modified by (Suleiman et al. 1996). Semen samples were centrifuged at 800x g for 10 minutes, and the supernatant was discarded. Then, the sperm pellet was resuspended in 1 mL of PBS (pH 7.2) or a variable volume of PBS to obtain a sperm concentration of 20×10⁶/mL. For reaction, to 2 mL of TCA-TBA reagent (5% (w/v) TCA; 0.375% (w/v) TBA in 0.25N HCl; in a ratio of 1:1:1), 1 mL of the above supernatant was added, and the mixture was heated in a boiling water bath for 15 min. After cooling, the suspension was centrifuged at 500 xg for 10 minutes, the supernatant was separated, and absorbance was measured at a maximum wavelength of 535 nm in a UV-VIS Spectrophotometer (DBS; Model-UV 3092, LAB INDIA). Similarly, a standard graph was plotted using HCl-digested 1, 1, 3, 3-tetraethoxy propane (1, 1, 3, 3-TEP), and after overnight digestion, an equimolar concentration of MDA was prepared. The MDA concentration was calculated by the specific absorbance coefficient of 1.56×10⁵/mol/cm3.

LPO (nM MDA/100 × 10⁶ sperm) = OD x 10 x test volume/1.56 x test volume.

Post-thaw sperm kinematics parameters

Motility and kinematic parameters of spermatozoa were assessed using a Computer-assisted semen analyzer (CASA), Minitube, Tifenberg, Germany, supported with AndroVision® software (Version 6.1). The CASA is equipped with a Zeiss Axioscope Microscope, Germany, with a 20X negative phase contrast with a blue filter to determine sperm motility and kinetics. The microscope has a thermostatic stage to maintain a constant temperature of 37°C. For motility and kinematics, at least ten fields were counted, having a minimum of 2000 spermatozoa. Total motility (TM,%), progressive motility (PM,%),curvilinear velocity (VCL,µm/s), straight-line velocity (VSL, µm/s), average path velocity (VAP,µm/s), the amplitude of lateral head displacement(ALH, µm/s), beat cross frequency (BCF, Hz), linearity (LIN), straightness (STR) and wobble (WOB) measured by CASA. The spermatozoa were classified as immotile when HAC (Head Activity) < 0.087; local motile when VCL < 48 and VSL < 24; circular motile when radius > 9 and < 90 and rotation > 0.70; slow motile when VCL < 120; fast motile when VCL > 120; and hypermotile when VCL > 150, LIN < 0.5 or 50% and ALH > 4.

Statistical analysis

The SPSS version 25 (IBM, Armonk, NY, USA) statistical package was used to conduct all the statistical analyses. Differences associated with p-values ≤ 0.05 were considered significant. As all the outcomes were continuous variables, data were reported as Mean ± SE in tables. Two-way analysis of variance (ANOVA) was used to assess the sperm function tests at the initial, pre-freeze, and post-thaw stages of cryopreservation. These sperm function parameters were sperm progressive motility,sperm viability, membrane integrity, HOS (Hypo-osmotic swelling) response, and sperm acrosomal integrity. Sperm LPO by malondialdehyde (MDA) and sperm kinematics parameters were assessed post-thaw stage.Tukey's post hoc test was used to compare means and identify significant changes between the treatment groups at the initial, pre-freeze, and post-thaw stages of cryopreservation. Repeated measures analysis of variance (Two-way ANOVA) was used to assess the significance of the difference in post-thaw motility among treatment groups at different time intervals (Post-thaw incubation test).

Functional sperm attributes

Table 1 shows the effect of an extender with trehalose and glycerol and block freezing at initial, pre-freeze, and post-thaw sperm quality. The initial, pre-freeze, and post-thaw progressive motility,sperm viability, HOSresponse, and acrosome integritywere significantly higher (p ≤ 0.05) in T2 than inC and T1 samples.

Post-thaw semen evaluation showed that mean values of the rate of lipid peroxidation were significantly lower (p ≤ 0.05) in the T2 sample, followed by T1 and control samples. A significant difference (p ≤ 0.05) in mean values of lipid peroxidation was observed in both treatment samples (T1 and T2).

Table 1

Effect of block freezing method on seminal attributes of Sahiwal bull spermatozoa at different stages of freezing (Mean ± SE; N = 48)
Seminal attributes	Stage of semen freezing	Control	Treatment-1 (T1)	Treatment-2 (T2)
Progressive motility (%)	Initial dilution	77.91 ± 0.59	77.91 ± 0.59	77.91 ± 0.59
	Pre-freeze	67.71^b ± 0.52	68.54^b ± 0.56	72.08^a ± 0.51
	Post-thaw	51.25^c ± 0.45	54.17^b ± 0.49	56.46^a ± 0.56
Viability (%)	Initial dilution	87.77 ± 0.49	87.77 ± 0.49	87.77 ± 0.49
	Pre-freeze	79.00^b ± 0.46	79.41^b ± 0.41	82.4^a ± 0.45
	Post-thaw	64.62^c ± 0.61	67.04^b ± 0.56	70.02^a ± 0.45
HOS response (%)	Initial dilution	70.56 ± 0.36	70.56 ± 0.36	70.56 ± 0.36
	Pre-freeze	62.69^b ± 0.41	64.10^a ± 0.41	65.23^a ± 0.44
	Post-thaw	54.79^c ± 0.54	56.79^b ± 0.57	59.37^a ± 0.65
Acrosome integrity (%)	Initial dilution	89.81 ± 0.37	89.81 ± 0.37	89.81 ± 0.37
	Pre-freeze	82.17^b ± 0.34	82.60^b ± 0.35	83.58^a ± 0.29
	Post-thaw	73.64^c ± 0.41	75.02^b ± 0.37	77.17^a ± 0.35
Rate of lipid peroxidation (nM MDA/100 million sperm)	Post-thaw	2.16^a ± 0.12	1.55^b ± 0.08	1.20^c ± 0.07
Values (Mean ± SE) bearing different superscripts (^a,b,c) in a row in each seminal attribute differ significantly (p ≤ 0.05). SE: Standard error of Mean, MDA: Malondialdehyde

Post-thaw incubation test

The mean post-thaw progressive motility percent at 0, 30, 60, 90, and 120 minutes were significantly higher (p ≤ 0.05) in the T2 than in the C and T1 samples. Values in the T1 were significantly (p ≤ 0.05) higher than the C samples. Both treatment samples (T1 and T2) differed significantly (p ≤ 0.05) from each other and C samples, as shown in Table 2.

Table 2

Post-thaw incubation test revealing post-thaw motility in Sahiwal bulls spermatozoa at different time intervals (Mean ± SE; N = 48)
Time (min)	Control	Treatment-1 (T1)	Treatment-2 (T2)
0	51.25^c ± 0.45	54.17^b ± 0.49	56.46^a ± 0.56
30	46.87^c ± 0.73	49.37^b ± 0.69	51.67^a ± 0.58
60	39.58^c ± 0.79	42.08^b ± 0.67	45.21^a ± 0.70
90	31.04^c ± 0.95	34.79^b ± 0.77	37.08^a ± 0.73
120	20.42^c ± 0.67	25.21^b ± 0.88	28.54^a ± 0.77

Means bearing different superscripts (^{a, b, c}) in a row differ significantly (p ≤ 0.05).

Post-thaw sperm kinematics parameters

CASA parameters such as TM (%), PM (%), VCL (µm/s), VSL (µm/s), VAP (µm/s), BCF (Hz), ALH (µm) were significantly higher (p ≤ 0.05) in T2 sample than C and T1 samples. There was no significant difference in LIN and WOB between the three samples. The value of STR was significantly higher (p ≤ 0.05) in the treatment groups (T1 and T2) than in the control group (Table 3).

Table 3

Effect of block freezing method on post-thaw sperm kinematic parameters (CASA) (Mean ± SE; N = 48)
CASA Parameters	Control	Treatment-1 (T1)	Treatment-2 (T2)
TM (%)	58.86^c ± 0.61	61.20^b ± 0.85	65.18^a ± 0.74
PM (%)	49.73^c ± 0.67	52.70^b ± 0.94	57.04^a ± 0.89
VCL (µm/s)	78.04^c ± 1.48	82.67^b ± 1.56	88.12^a ± 1.91
VSL (µm/s)	31.84^c ± 0.59	33.93^b ± 0.57	35.58^a ± 0.78
VAP (µm/s)	39.85^c ± 0.71	42.10^b ± 0.70	44.15^a ± 0.93
BCF (Hz)	9.35^b ± 0.18	9.79^b ± 0.20	10.62^a ± 0.18
ALH (µm)	0.88^b ± 0.01	0.91^b ± 0.02	0.98^a ± 0.02
STR	0.79^b ± 0.003	0.81^a ± 0.002	0.81^a ± 0.002
LIN	0.41 ± 0.003	0.41 ± 0.003	0.40 ± 0.003
WOB	0.51 ± 0.002	0.51 ± 0.003	0.50 ± 0.002

Means bearing different superscripts (^{a, b, c}) in a row differ significantly (p ≤ 0.05).TM: Total Motility, PM: Progressive Motility, VCL: Curve Linear Velocity, VSL: Straight Line Velocity, VAP: Average Path Velocity, BCF: Beat Cross Frequency, ALH: Amplitude of Lateral Head Displacement, STR: Straightness, LIN: Linearity, WOB: Wobble

The study compared the advantages of trehalose incorporation and modification in the freezing protocol with traditional freezing to understand the post-thaw functional characteristics of bovine sperm. The findings revealed that block freezing with 25% trehalose (T2) and 5% glycerol significantly (p ≤ 0.05) enhanced the post-thaw sperm quality.Along with increased viability and intact membranes in spermatozoa, improvements in sperm kinematics and motility characteristics were also noted. MDA concentrations decreased, suggestingblock freezing causes less oxidative stress to spermatozoa than traditional freezing.

Studies have reported that trehalose improves sperm motility, viability, morphology, acrosome integrity, DNA integrity, and mitochondrial functionality in fast freezing (Hu et al. 2009; Schulz et al. 2017). This is consistent with our study because we also performed fast freezingby reducing equilibration and vapour freezing time.

Also, to capture the latent heat released by the freezing process, a heat-conductive aluminium metal block was used in our study, which might have improved the sperm parameters. Several sugars have also been used to freeze spermatozoa quickly. Sucrose is the most frequently used carbohydrate for the quick freezing of sperm in Humans(Isachenko et al. 2008), Dog (Sanchez et al. 2011), Donkeys (Diaz-Jimenez et al. 2018)and Mouflons and Ibex (Boveda et al. 2018). Other carbohydrates, like trehalose and raffinose, have recently been investigated for rapid sperm freezing in dogs (Caturla et al. 2018) and mice (Horta et al. 2017). In a study, 0.25 mL straws were used to find the ideal concentration, volume, and kind of carbohydrate for vitrifying sperm in stallions. They discovered that trehalose preserved sperm functional characteristics better (Consuegra et al. 2019).

Studies regarding the comparison between trehalose with different sugars (fructose, galactose, glucose, xylose, lactose, maltose, sucrose, mannose, and raffinose) and also with some other additives (taurine, cysteamine, and hyaluronan) have been done in various species such as in bull (Woelders et al. 1997), ram (Bucak et al. 2007), goat (Khalili et al. 2009), Stallion (Squires et al., 2004), Dog (Yildiz et al. 2000), Red Deer (Fernandez-Santos et al. 2007), Humans (Isachenko et al. 2003), and Mouse (An et al. 2000).All of these studies found that trehalose preserved the sperm parameters better. Trehalose is an excellent protein stabiliser and aids in maintaining the activity of enzymes both in solution and when they are freeze-dried as reported by Kaushik and Bhat (2003). It lowers the heat capacity of protein denaturation and raises the transition temperature, leading to better protein stability.At lower concentrations, trehalose has been shown to improve sperm vitality in several studies (Sanchez et al. 2011;Chhillar et al. 2012; Aisen et al. 2005)which supports the present study's findings. It has been used in various studies as vitrification diluent (Hu et al. 2009; Schulz et al. 2017; Consuegra et al. 2019). Because our study also did quick freezing by lowering equilibration and vapour freezing times, we used trehalose in the diluent to accomplish block freezing in our study. This combination may have resulted in more effective cooling rates, less mechanical damage, and more appropriate dehydration-rehydration in sperm cryopreservation.Another reason for improved semen parameters in the present study's treatment group could be the lower concentration of glycerol (5%) used in it compared to the control group (6.4%). Glycerol, a common cryoprotective agent in semen cryopreservation, can have cytotoxic effects on sperm at varying concentrations. Studies indicate that glycerol concentrations can impact sperm motility, viability, and abnormalities in different species. Studies on rooster semen (Wu et al. 2020) and Bali cattle sperm reported by Lestari and Rosadi (2022) highlights the importance of glycerol concentration in maintaining sperm quality during cryopreservation. While lower concentrations, like 5%, are beneficial for sperm motility and viability in stallions (Azizah 2009), higher concentrations can lead to increased abnormalities and decreased motility in Bali cattle as reported by Lestari and Rosadi (2022).

In a study, a synergistic effect of 5% glycerol with 100 mmol trehalose and 7% glycerol with 50 mmol trehalose was found to increase sperm viability significantly than other combinations or glycerol alone in Ram (Najafi et al. 2013), which is similar to our study. Another study found that adding 3 or 5% glycerol combined with 100 mmol trehalose to the extender increased the vitality of ram spermatozoa (Azizah 2009; Najafi et al. 2013).

In the present study, sperm progressive motility at the initial, pre-freeze, and post-thaw stage of cryopreservation was significantly (p ≤ 0.05) higher in T2 group (25% trehalose and 5% glycerol in TFEG extender combined with block freezing) than control (6.4% glycerol with no trehalose in TFEG extender combined with conventional freezing) and T1(25% trehalose and 5% glycerol in TFEG extender combined with conventional freezing) group. This is consistent with various studies in which trehalose in extenderhelps to enhance sperm progressive motility (Chhillar et al. 2005; Reddy et al. 2010; Shaikh et al. 2016; Suksai and Dhanaworavibul (2019); Al-Badrany et al. 2020)when used in semen extenders. This positive effect could be attributed to the fact that trehalose causes minimal changes in the osmolarity of the extender compared to other sugars (Gao et al. 1995).These results are consistent with earlier studies conducted on bull semen(Hu et al. 2010; El-Sheshtawy et al. 2015; Ghareeb et al. 2017; Al-Badrany et al. 2020).

Evaluation of sperm viability showed a significant (p ≤ 0.05) improvement after freezing-thawing in T2 compared to other samples (C and T1), and our results are consistent with previous studies (Buffalo Bull: Reddy et al. 2010; Bull: Chhillar et al. 2005; Shaikh et al. 2016; Equine: El-Badry et al. 2017; Human: Suksai and Dhanaworavibul (2019). In our study, we found that trehalose was more effective at preserving sperm membrane integrity at a low concentration,similar to other studies(Chhillar et al. 2005; Reddy et al. 2010; Shaikh et al. 2016; El-Badry et al. 2017). However,a study in goats done by Aboagla and Terada (2010) revealed that extending sperm with (100%) trehalose increased sperm membrane fluidity, rendering the sperm capable of enduring freeze-thaw damage which is contradictory to our study.This may be due to the higher concentration of trehalose used in this study. The possible explanation behind the improved sperm viability in our study could be that trehalose is a non-permeable cryoprotectant. It increases the solute concentration extracellularly, and dehydrates the sperm cells by dragging water out. By this it ultimately helps in reducing the production of intracellular ice crystals.

In addition, block freezing conferred lower damage to the sperm membrane due to less ice crystal formation. The use of highly heat conductive aluminium blocks in the present experiment might be assisted in capturing the latent heat release during freezing and minimised the freezing medium's rewarming during the temperature drop from − 5 to -15°C. It has been claimed that freezing occurs from the periphery to the center and that when it does, latent heat is released, which travels to the ice front, causing sperm to freeze and thaw repeatedly as reported by Arav and Saragusty (2016).

In the present study, HOS's response was significantly (p ≤ 0.05) higher in the T2 group than in the T1 and control group at the initial, pre-freeze, and post-thaw stages of semen cryopreservation. The first reason behind this could be because trehalose interacts with phospholipids in the plasma membrane, increasing its membrane fluidity, which makes spermatozoa more resistant to damage from freeze-thawing. This interaction may account for higher HOS response. The second reason could be that trehalose preserves biomolecular structures by trapping necessary water molecules for hydration as reported by Patist and Zoerb (2005) and substituting water in hydrogen bonds (Chen et al. 2000). It is an excellent and exceptional protein stabilizer in many arctic species. It increases the transition temperature of proteins and decreases the protein denaturation temperature, which causes the flattening of the stabilisation curve and increases the free energy of stabilisation.

The third reason for the higher HOS response in the present study may be that block freezing was performed rather than conventional freezing, which induced less stress on the sperm by lowering the temperature from 4°C to -196°C at a faster rate without supercooling.The optimum cooling rate plays a vital role in maintaining the structural integrity of spermatozoa. Higher cooling rates at 2 cm in the current study allowed spermatozoa to pass through the risky area faster, lowering ice crystal formation and minimising spermatozoa damage (Ruilan 2018; Zhang et al. 2024). The last reason may be that the concentration of glycerol used in the present study is 5% less than that used in conventional freezing (6.4%). Various studies have shown that the cytotoxic effects of glycerol and the reduced concentration used in the present study may be another reason for more HOS-responsive sperms.

A study was reported in which semen that had been extendedwith tris glucose, citric acid, and glycerol when exposed to liquid nitrogen vapours for varied amounts of time (5, 10, 15, and 20 minutes) and at varying heights of straws from the vapours (2 cm, 4 cm, 6 cm, and 8 cm) (Zhang et al. 2024). Most semen characteristics, including progressive motility, viability, and acrosome integrity, were better preserved after 20 minutes of vapour freezing at the height of 2 cm from liquid nitrogen,which isconsistent with our study.

Our study found that sperm acrosome integrity was better preserved in the T2 group than in the control and T1 groups. This is consistent with findings from other studies(Chhillar et al. 2005; Azizah 2009; Al-Badrany et al. 2020), which found that trehalose preserves acrosome integrity more effectively at lower concentrations. Research on Dogs(Yildiz et al. 2000)and Boars(Gutierrez-Perez et al. 2009; Hu et al. 2010) shows that trehalose helps minimize acrosomal damage during cryopreservation.

A post-thaw incubation test revealed that after 4 hours,trehalose preserved sperm motility better than a control containing no trehalose, consistent with a study done in rams (Azizah 2009). This may be due to trehalose's exceptional stabilization action, which better preserved sperm motility even after 4 hours.

In the present study, the rate of lipid peroxidation was significantly lower (p ≤ 0.05) in the T2 sample, followed by T1 and control samples. There have been numerous studies claiming that trehalose supplementation significantly increases catalase and gluthathione peroxidise activity in Bull (Hu et al. 2009; Chhillar et al. 2012; Shaikh et al. 2016), Buffalo (Reddy et al. 2010; Badr et al. 2010), and Ram (Aisen et al. 2005; Bucak et al. 2007 ) sperm which is in agreement with the present study. As a result of the semen's exposure to cold shock and atmospheric oxygen during cryopreservation, which increases the generation of reactive oxygen species, the sensitivity to lipid peroxidation is increased (Perumal et al. 2011). Lipid peroxidation, which can result in reduced sperm motility or cell death, involves free radicals. Trehalose was thus added to the semen in the present research since it has antioxidant qualities and may help prevent the production of ROS, which would otherwise harm the spermatozoa (Uysal et al. 2007).

Trehalose supplementation improved the post-thaw sperm kinematic parameters in buffalo bulls (Iqbal et al. 2018) and Angora buck (Tuncer et al. 2011). This is inconsistent with our study, in which trehalose at a lower concentration (25%) preserved the post-thaw CASA parameters of sperm better than at a higher concentration (50%). These results may be because trehalose acts as a cryostabilizer of sperm membrane proteins and modifies the shape of ice crystals, which ultimately preserves sperm motility.In conclusion, the block freezing protocol using 25% trehalose and 5% glycerol substantially improved sperm functional parameters after freezing-thawing compared to conventional freezing. Block freezing shortened the time required for cryopreservation by employing a shorter equilibration and vapour freezing time. The combination of cryoprotectants (trehalose and glycerol) enabled quick freezing in the form of block freezing.The measurement of LN₂ vapour temperature at a distance of 2cm from straws, as well as field fertility, are areas that can be investigated further.

Contributions

ManishaSethi conceived and designed the study under the guidance of Tushar Kumar Mohanty and MukeshBhakat. ManishaSethi, Nadeem Shah and Dileep Kumar Yadav conducted the research experiment, data analysis. ManishaSethi and Nadeem Shah interpreted the findings and wrote manuscript under the guidance of Dilip K Swain. Tushar K Mohanty, and MukeshBhakat proofread the manuscript after it was finalized by Rubina K Baithalu and Nishant Kumar. All authors reviewed the results and approved the final version of the manuscript.

Acknowledgments

Director, ICAR-National Dairy Research Institute, Karnal, Haryana (India) for providing the facility to conduct the research work and acknowledged the financial support of XII Plan Scheme-Incentivizing Research in Agriculture, Project V: Semen sexing in cattle (No. CS/F.No.16-8/2017-1A.IV Dt.26.10.2017).

Funding

This research was supported financially by XII Plan Scheme-Incentivizing Research in Agriculture, Project V: Semen sexing in cattle (No. CS/F.No.16-8/2017-1A.IV Dt.26.10.2017).

Data availability

All data are available within the manuscript

Statement of Animal Rights

Institutional Animal Ethical Committee approved all experimental protocols and semen collection using artificial vaginas (45-IAEC-19-4).

Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

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Tris and Trehalose based modified cryopreservation dilutor in block freezing improves cryo-survivability and functionality of Sahiwal bull semen

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Abstract

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Introduction

Materials and Methods

Chemicals and reagents

Experimental bulls and semen collection

Semen equilibration and freezing

Post-thaw evaluation of the semen

Assessment of sperm progressive motility

Assessment of membrane integrity

Assessment of sperm acrosomal integrity

Assessment of sperm lipid peroxidation by malondialdehyde (MDA) estimation

Post-thaw sperm kinematics parameters

Statistical analysis

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Functional sperm attributes

Post-thaw incubation test

Post-thaw sperm kinematics parameters

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