Effect of Extender Supplementation With Caffeine on Arabian Stallion’s Semen Quality After Freezing

In the current study, we aimed to investigate the effect of adding different levels of caffeine to the diluent on the Arabian stallion’s sperm quality post cryopreservation. Semen cryopreservation is an essential part of artificial insemination in Arabian horses. In most of cases, the stallion selection is based on desirable phenotypic and physical traits rather than on fertility. This approach may have resulted in the propagation of low-fertility genotypes that are unable to withstand the various stresses associated with cryopreservation such as freezing-thawing stress, oxidative stress


Introduction
Artificial insemination in horses has long been used worldwide to propagate the superior stallions (Ropka-Molik et al., 2019;Cosgrove et al., 2020).Semen cryopreservation is the key factor for artificial insemination success and allow preserve the genetics of a stallion for boundless time.However, equine genetic selection depends mainly on favourable phenotypic traits regardless of fertility (Morillo-Rodríguez et al., 2012;Carneiro et al., 2018).Subsequently, some stallions are vulnerable to different stressors present during semen cryopreservation.Accordingly, mitigation strategies are needed to enhance semen cryopreservation process in stallions.The stressors during cryopreservation process include; freezing-thawing stress, oxidative stress and osmotic stress.These stressors are harmful to sperm cell and negatively affects plasma and acrosome membranes, structure and function of spermatozoa survival and depletion of ATP and mitochondrial membrane potential and cause lipid peroxidation (Gibb & Aitken, 2016;Irato & Santovito, 2021).To protect spermatozoa from these fatal stressors, it is necessary to extend the semen during long storage at extremely low temperature (-196 °C) in appropriate diluents include certain substrates to avoid harmful effects of cryogenic injury on spermatozoa.Numerous buffers and cryoprotectants have been used for semen to increase post-thaw sperm quality (Coyan et al., 2010;Alamaary et al., 2019;Nikitkina et al., 2020).One of the major causes of the different stressors is the excessive formation of reactive oxygen species (ROS) (i.e., mainly H 2 O 2 and NO 2 -) which damage the integrity of the sperm cells, resulting in low fertilization (García et al., 2017;Pisoschi & Pop, 2015).The balance between ROS formation and antioxidants in the sperm cell is crucial for capacitation, hyperactivation and acrosome reaction (O'Flaherty et al., 2006).However, when imbalance happens between ROS production and antioxidant levels, oxidative stress occurs, accordingly, spermatozoa quality decreases (Castro et al., 2016).Various antioxidants (enzymatic or non-enzymatic) have been used in equine to overcome the negative effects of excessive ROS formation (Gibb & Aitken, 2016;Del Prete et al., 2019;Irato & Santovito, 2021).
Caffeine is a good candidate antioxidant to be involved in semen extender to overcome the negative effects of oxidative stress and other accompanying stressors (Azam et al., 2003;Patel & Sigman, 2008;Carocho & Ferreira, 2013;Stephens et al., 2013;Agarwal & Majzoub, 2017;Halo et al., 2022Halo et al., , 2023)).Caffeine could elevate the levels of cyclic AMP in the sperm cell because of its properties as phosphodiesterase inhibitor.As cyclic AMP increases, glycolysis accelerates results in abundant ATP formation which is crucial for spermatozoa motility and hyperactivation (Stephens et al., 2013;Halo et al., 2023).Caffeine is a potent antioxidant, which could contribute to the overall antioxidant's capacity of caffeine-containing diluents that may lead to improve spermatozoa motility and fertilizability (Azam et al., 2003).Caffeine is capable to protect stallion spermatozoa from oxidative stress (Halo et al., 2022).
Therefore, the aim of the present study is to evaluate the role of caffeine when supplemented to the basic extender (HF-20) on the post-thaw spermatozoa quality parameters of Arabian stallions.

Animals
In the current experiment five stallions between 4 to 10-year old will be used for semen collection.All stallions were healthy and examined for breeding soundness.The stallions were kept individually in an open barn and offered pellets, alfalfa hay, clean water, and integrated mineral licks.Semen collections is done twice weekly from each stallion using artificial vagina and estrus-mare.

Semen Collection and Processing
Upon semen collection, the gel is removed immediately from the semen and then transferred to a water bath (37 °C).The semen volume is recorded.The ejaculates are evaluated for general progressive motility and sperm concentration.Sperm concentration and motility were determined using the CASA system (ISAS program, Prosser R+D, Paterna, Valencia, Spain).Samples with a minimum concentration of 200 × 10 6 sperm/ml and motility > 60% used for this study.
The filtered semen of each ejaculate was diluted (1:1) with a centrifuged medium and then divided into 4 aliquots.The aliquots were centrifuged at 800 g for 10 minutes, the seminal plasma was removed, and each sample was re-suspended with FH-20 (0) without any supplementation as a control.HF-20 extender is supplemented with caffeine at a concentration of 0.5 mM, 1 mM, 3 mM and 5 mM.The final semen concentration after dilution was 200 × 10 6 sperm/ml.All of the tubes were cooled to 4 °C for 90 minutes before being assessed for motility, morphology, and sperm membrane integrity.Cooled semen is filled into 0.5 ml straws.The straws were frozen by putting straws horizontally on the liquid nitrogen surface (9 cm) for 9 minutes and then plunged in liquid nitrogen.

Extenders
The centrifuged medium was a mixture of 6.0 g glucose, 0.37 g ethylene-diamine-tetra-acetic acid (EDTA), 0.37 g sodium citrate, 0.12 g sodium bicarbonate, 100,000 IU penicillin, and 0.08 g streptomycin in 100 ml of distilled water.HF-20 as freezing extender is used.
Freezing extender with egg yolk was prepared by dissolving 5.0 g glucose, 0.3 g lactose, 0.3 g raffinose, 0.15 g sodium citrate, 0.05 g sodium phosphate, 0.05 g sodium potassium tartrate, and 3 mL glycerol in 50 mL of distilled water and EY at 10 mL is added as a basic buffer, and then distilled water is added to a total volume of 100 mL (control extender).Caffeine at different concentrations (0.5, 1, 3 and 5 mM) is dissolved in the basic buffer.In all cases, the pH of the extender was adjusted using sodium bicarbonate buffer.Measurements of pH were adjusted using a pH meter (Hanna, model HI-2212, Woonsocket, RI, USA).

Cryopreserved Semen Evaluation
The frozen straws were thawed in a water bath at 38 °C for 60 seconds, and the contents were expelled into a small warm tube.The general and progressive motility were then evaluated using the ISAS program and assessed for plasma membrane integrity, morphology defects, acrosome integrity, and viability.Frozen semen evaluation is conducted at the AI laboratory for Animal Production and Breeding Department, College of Agriculture, Qassim University.

Assessment of Sperm Motility
The ISAS program (CASA system) is used to assess the motility patterns immediately after the dilution of the semen or the post-thawed semen.A sample (2.7 μL) from each tube is placed onto a slide and semen motility is assessed based on five digital images from different fields via a × 10 negative-phase contrast objective and warm stage at 38 °C.Motility patterns are measured according to the total motile sperm (TMS %), rapid progressively motile (RPS %), curvilinear speed (VCL μm/s), rectilinear speed (VSL μm/s), average value (VAP μm/s), linearity index (LIN %), and straightness index (STR %).At least 300 sperm are analyzed from each sample, and the images are read within one second.

Plasma Membrane Integrity
A hypo-osmotic swelling test (HOST) was used to assess the plasma membrane integrity of the spermatozoa.A minimum of 100 sperm cells are analyzed for the coiled tail using phase contrast microscopy (× 400).A mixture of a glucose-based solution at 100 mOsmol and 20 μL of semen is incubated at 37 °C for 50 minutes in a water bath (Neild et al., 1999).

Viability Test
Sperm viability is evaluated using an acridine orange (AO) and propidium iodide (PI) kit (Halotech DNA S.L., Madrid, Spain).First, the semen is diluted to 10-15 × 10 6 sperm/ml.Then 10 μL of diluted semen is placed on a slide.Then 1.0 μL of AO and PI is mixed with the diluted semen.Finally, the mixture is covered and then evaluated using a fluorescence microscope.Living sperm retained the AO, producing green fluorescence, while PI penetrated the damaged sperm, causing red fluorescence.A total of 300 sperm are assessed per sample.

Morphology
Sperm morphology is examined using the Hancock's solution.A 15 μL drop of semen is mixed with 15 μL of in a clean and warm tube.The mixture is dropped and covered, and evaluated under oil immersion at 1000 × magnification.The spermatozoa's morphological defects are classified sperm abnormalities according to their effect on fertility: major defects include most abnormalities of the head and midpiece, proximal cytoplasmic droplets and single abnormalities present in a high percentage, whereas minor defects include looped tails, detached sperm heads, and distal cytoplasmic droplets (Blom, 1983).

Acrosome Integrity
A drop of semen sample is mixed with a drop of isotonic Hancock's solution with 6% of formaldehyde on a clean and warmed microscope slide.The mixture is covered and assessed in a light microscope at x1000 at least 200 sperm for acrosome defects.

Results
The after-cooling results are represented in Table 1.The results showed that extender supplemented with 1 mM Caffeine was the best in progressive motility 51.55±4.12% vs. 31.66±8.96% in control (p < 0.05).However, there were no significant effects on total motility in concentrations 0.5, 1 and 3 mM caffeine.As caffeine supplementation increased to 5 mM the total motility significantly dropped from 85.00±2.88% in control group to 63.33±2.35%(p < 0.05).The 0.5 and 1 mM caffeine extenders were the highest in VCL, VSL and VAP (p < 0.05).The morphological defects were higher in 1 and 3 mM caffeine extenders (p < 0.05).The results of HOST showed that the elevated concentration of caffeine (5 mM) were the lowest compare to control (53.63±5.25 vs. 75.87±5.29,respectively) (p < 0.05).The post-cryopreservation results are represented in Table 2.The results showed that extender supplemented with 1 mM Caffeine was the best in progressive motility 26.45±2.85% vs. 16.73±2.02%in control (p < 0.05).
The results of TM showed that extender supplemented with either 0.5 or 1 mM caffeine had the highest TM (37.50±1.44 and 48.75±3.14, respectively) compared to control (26.66±3.33)(p < 0.05).However, there were no significant effects on VCL and VAP.The addition of caffeine at any concentration studied improved the VSL, LIN, STR and HOST (Table 2) (p < 0.05).The extender supplemented with 0.5 mM caffeine was the highest in morphological defects and the lowest in acrosome integrity (p < 0.05).The vital sperms % was the highest (67.72±5.23%)compared to 42.71±11.54%in control (p < 0.05).

Discussion
The main problem of preserving spermatozoa at very low temperature is the cryoinjury which hindered the quality and motility of cryopreserved spermatozoa.Extenders are the most important part in semen cryopreservation as they have the required components to protect spermatozoa from cryoinjury and maintain motility and viability.
During cryopreservation, reactive oxygen species (ROS) are generated and could damage the spermatozoa due to oxidative stress (Agarwal et al., 2008;de Lamirande et al., 2008, Sun et al., 2020).The lipid peroxidation of spermatozoa increases during semen preservation procedures, especially in the spermatozoa midpiece.It is hypothesized that caffeine supplementation could improve the quality parameters and viability of stallion sperms during the cryopreservation, which is crucial for improving the number of viable and fertilizable sperms post freezing.The present study showed that 1 mM of caffeine is desirable to sustain sperm motility and viability post freezing.
The generated ROS are responsible for sperm cell organelles damages, DNA fragmentation, and hence inferior motility, which negatively affects the cryopreservation process.Therefore, the inclusion of certain antioxidants during dilution could protect the cryopreserved spermatozoa through prevention of ROS generation (Tvrdá et al., 2011).
In the current research, caffeine supplementation at low concentrations improves cryopreserved sperms motility and viability.One of the possible positive effects of caffeine is the activation of cAMP production (Chavda et al., 2022).
Our results showed that low doses of caffeine significantly improve the total and progressive motility post-thawing.This is maybe partially supported by the work of Ramirez-Perez et al. (2022).However, we used only caffeine in our study.Other studies supported the importance of antioxidants during cryopreservation on spermatozoa (Alomar, 2021;Shehab-El-Deen et al., 2021, 2023).The addition of caffeine post-thawing maybe needed to support the caffeine we added during cryopreservation process, whereas, Rota et al. (2019) reported that the post-thawing addition of caffeine improved the motility of sperms.

Conclusions
From the study results, it could be concluded that 1 mM caffeine concentration is suitable additives to Arabian stallion semen diluters.Caffeine supplementation at low concentrations improved motility and viability parameters of Arabian stallion spermatozoa post cryopreservation.