Enhancing liquid-chilled storage and cryopreservation capacities of ram spermatozoa by supplementing the diluent with different additives

Objective In the present study, we determined efficiency of incorporating caffeine, melatonin or omega-3 polyunsaturated fatty acid in the diluent on mitigating consequences of (a) liquid chilled- and (b) cryo-storage of ram spermatozoa. Methods In the first experiment, ejaculates (n = 30) were collected from 5 adult rams and were pooled, diluted (1:10) with Tris-citric acid (base diluent) and were split into 4 aliquots assigned for: control (untreated), caffeine (0.1 mM), melatonin (0.3 mM) or omega-3 fatty acids (0.3 mM) (T0). The diluted specimens were stored at 4°C for 48 h, during which sperm physical and cytological properties were evaluated along with oxidative stress indices (T24, T48). In the second experiment, 15 ejaculates (3 per male) were pooled, diluted with glycerolized base diluent (4% glycerol, v/v) and were split corresponding to the same previous treatment groups before being processed for cryopreservation. Post-thaw physical and kinematic sperm properties were assessed by a computer-assisted sperm analysis system. Results The results clarified superiority of both melatonin and omega-3 supplementation on maintaining (p<0.05) sperm properties, while reducing (p<0.05) lipid peroxidase reaction and enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in preservation medium, compared to caffeine either during liquid-chilled storage or cryopreservation of spermatozoa. Conclusion Melatonin and omega-3 are regarded efficient alternatives to caffeine when processing ram spermatozoa for application of artificial insemination or in vitro fertilization.


INTRODUCTION
Extensive application of sophisticated reproductive techniques played a vital role in improv ing livestock industry in recent years. Artificial insemination (AI) and in vitro fertilization (IVF) are among the most applicable techniques utilized to achieve this goal in large do mestic animals. In sheep, however, successful application of such technologies is still lacking although it is essentially required to develop breeds with superior productivity, resistant against diseases, or high capacity to cope with harsh environmental conditions [1]. Con sequently, developing sperm processing procedures, to maintain sperm physical properties and fertilization potential, became a prerequisite for successful application of these tech niques.
Liquidchilled storage is considered most appropriate for sperm preservation when se men is required within a short period of time after collection for AI or IVF [2]. However, the full potential of semen utilization, mostly in commercial application schemes, relies on cryopreserved doses [3]. Nonetheless, both sperm preser vation techniques have detrimental effects on physical and morphometric properties of spermatozoa and, hence, on sperm fertilizing capacity [4].
Sheep sperm cell membranes contain higher amounts of polyunsaturated fatty acids (PUFAs) than in other species [5]. Therefore, ram spermatozoa are highly susceptible to the oxidative damage that occurs during chilled storage [6] or freezing/thawing cycle of semen [7].
Primarily, the seminal fluid contains a wide spectrum of endogenous enzymatic and nonenzymatic antioxidants that act as free radical scavengers to protect spermatozoa [8]. However, the protective effects of such naturallyexisted antioxidants decrease due to dilution of semen for processing [9]. There fore, studies on inclusion of different antioxidants in sperm preservation medium became in focus of recent fertility related practices. In vitro supplementation of melatonin [10], caffeine [11] and n3 PUFAs [12] in the diluent have been reported to improve sperm preservation capacity in buffalos, rams and cow bulls, respectively. However, the degree of improvement varied dramatically among the studies due to species differences, as well as level and type of the supplement.
In the current study, two experiments were conducted to evaluate influence of incorporating melatonin, caffeine or omega3 PUFAs in the diluent on ameliorating the oxidative stress occurring during liquid chilled and cryopreservation of ram spermatozoa.

Ethics statement
All procedures were conducted conforming to the ISO 9001: 2015 quality management regulations and were approved by the Animal Care and Use Committee of Desert Research Cen ter, Egypt, complying with the guidelines and regulations of the Animal Ethics Committee Institute of the European Par liament for protection of experimental animals (2010/63/EU).

Animals
This investigation was implemented at the Artificial Insemi nation Lab., Mariout Research Station (Latitude 31° 00′ N; Longitude 29° 47′ E), Desert Research Center, Egypt. Five sexually mature Barki rams aged 36 to 48 months, and an aver age body weight of 45.0±2.0 kg, were used during April, 2017. All rams were housed in a fenced stockyard throughout the period of the study, and were allowed a daily grazing period from 0800 to 1400 h. Thereafter, they were fed a concentrate mixture according to their protein and energy requirements [13]. Egyptian clover, Trifolium alexandrinum, hay was pro vided ad libitum, and fresh water was presented once daily after returning from the pasture. Before executing the experi ment, all rams were clinically examined and were found free of disease or reproductive disorders.

Liquid-chilled storage medium (base diluent)
A Triscitric acid egg yolk base diluent was prepared for liquid chilled storage of ram spermatozoa as previously reported [14]. The diluent was clarified from egg yolk particles by cen trifugation at 2,400 g for 15 min and aspiration of the clear supernatant. The clarified base diluent was prepared 24 h prior to each collection session and was stored at 4°C until use.

Semen collection
Semen was collected 3 times weekly at 0700 h by an artificial vagina. Collection tubes with modified plastic water jackets were used to maintain the ejaculates at 37°C during the col lection sessions. Soon after collection, each raw ejaculate was transported to the laboratory, directly adjacent to the collec tion area, and was immediately evaluated for sperm physical and morphometric traits.

Raw ejaculates assessment and pooling
The raw ejaculates were kept in a warm water bath adjusted at 37°C throughout the assessment. Ejaculate volume (mL) was recorded using the graded collection tubes. The pH, sperm concentration (×10 6 /mL), mass motility score (5 = highly motile, 0 = immotile), progressive motility (%), viability (%), normal sperm (%), and intact acrosome (%) were also ana lyzed for each raw ejaculate. Accordingly, adequate ejaculates of each collection session from the same rams were pooled. Mean values of the aforementioned criteria in rawpooled ejaculates, throughout the period of the study, are displayed in Table 1.

Experimental design
Effects on oxidative status and liquid-chilled storage capacity of spermatozoa: In this experiment, 30 ejaculates were ob tained from the 5 rams, 6 ejaculates each, and ejaculates of each collection session were pooled and diluted (1:10) with the clarified base diluent. The diluted specimens were further split into 4 aliquots. The first aliquot served as control (un  [14]. The later adjunct consisted of 52.6% eicosapentaenoic acid (1.65 mmol/L EPA, 20:5 n3), 26.3% docosahexaenoic acid (0.76 mmol/L DHA, 22:6 n3) and 21.1% αlinolenic acid (0.72 mmol/L ALA, 18:3 n3). The omega3 oil was emul sified in the egg yolkcontaining base diluent and was vortexed for 2 min prior to the dilution process assuring that the oil was properly mixed with the medium. Immediately after di lution (T 0 ) all specimens were transported to a cooling cabinet (4°C) and were stored for 48 h, during which sperm physical and morphometric properties were evaluated along with oxi dative stress indices at 24 h interval (T 24 , T 48 ).

Semen assessment
Total sperm motility (%) was evaluated using a phasecontrast microscope (Leica Inc., Wetzlar, Germany) at 40× magni fication, whereas viability was assessed by eosinnigrosin differential staining technique at 1,000× magnification. Ro manowski's triplestain method (DIFFQUICK III, Vertex, Cairo, Egypt) was used to evaluate primary and secondary sperm abnormalities, as well as acrosomal cap integrity. Smears preparation and staining processes were performed as per the manufacturer' s instructions, and stained smears were evaluat ed by a phasecontrast microscope at 1,000× magnification. The functional integrity of sperm plasma membrane was determined by the hypoosmotic swelling (HOS) test [17], where at least 200 sperm were evaluated at 40× magnification.

Determination of oxidative stress indices and enzymatic activities
A portion of each semen group (2 mL) was aspirated and centrifuged (1,000 g for 10 min) at times parallel to those of sperm assessment (T 0 , T 24 , and T 48 ). The aspirated supernatant was stored at -20°C until oxidative stress indices and enzy matic activities were analyzed. The changes in total antioxidant capacity (TAC), malondialdehyde acetate (MDA) concentra tion, reduction of the resazurin dye test (RRT) and alkaline phosphatase (ALP) activity were analyzed by colorimetric kits (Biodiagnostic, Cairo, Egypt). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were analyzed colorimetrically by kits obtained from Spectrum, Egypt. All procedures were conducted according to the manu facturers' instructions.

Effects on cryopreservation capacity of ram spermatozoa
Cryopreservation medium and semen processing: After cen trifugation and clarification as illustrated previously, the base diluent was supplemented with 2% glycerol at 37°C, and was split into 4 aliquots representing the same previously men tioned additives. Another fifteen ejaculates were collected from the same 5 rams, in 3 collection sessions, and, similar to that of experiment 1, ejaculates of each collection session were pooled and diluted (1:10) with the glycerolized base diluent (portionA, T 0 ). All specimens were equilibrated for 3 h at 4°C. Thereafter, a second aliquot of chilledglycerolized dilu ent (portionB, T 3 ) was added to portionA to reach a final concentration of 4% glycerol in whole medium. Afterwards, the specimens were equilibrated for another 2 h at 4°C (T 5 ) before being packed in 0.5 mm French straws (200×10 6 sperm/ straw) using a minitübe filling and sealing machine (Model 133, Minitübe, Germany). The straws were placed in a mini tübe biological freezer, and were exposed to nitrogen vapor (-80°C) for 10 min before being immersed in liquid nitrogen. The frozen straws were stored under liquid nitrogen surface (-196°C) until physical and kinematic properties of sperma tozoa were analyzed by a computerassisted sperm analysis (CASA) system.

Statistical analyses
The data was checked by ShapiroWilk's test and were found fitting the normal distribution. Mean values of pooled (raw) sperm properties were obtained by simple ttest. The changes in the same sperm criteria, as well as oxidative stress indices and enzymatic activities, in liquidchilled specimens were analyzed by repeated measures analysis of variance (ANOVA) where the fixed effects of treatment, time (T 0 , T 24 , and T 48 ) and treatment by time interaction were determined. Further more, oneway ANOVA (F test) was used to compare CASA derived physical and kinematic sperm properties among control and treated groups. The statistical significance threshold was set at 5% and the differences between means were de tected by Tukey's posthoc test. The data were analyzed using IBMSPSS statistics program for windows [19]. The results are expressed as means±standard error of mean.

Effects on liquid-chilled storage capacity of ram spermatozoa
The results showed that the percent of progressive motility decreased (p<0.05) over time of storage in all groups. How ever, at T 48 , both melatonin and omega 3 supplemented groups recorded the highest (p<0.05) values of progressive motility (%) compared to control and caffeinesupplement ed specimens (Table 2). A similar trend was also observed in both percentages of live and normal spermatozoa ( Table 2). On the other hand, no significant difference was observed in the percent of primary sperm abnormalities among control and treated groups over the storage period. However, the per cent of secondary sperm abnormalities were higher (p<0.05) in control and caffeinesupplemented groups at T48 compared to melatonin and omega 3supplemented groups ( Table 2). It is worth mentioning that the observed secondary abnor malities comprised presence of distal/translocating cytoplasmic droplets with subsequent tail opening and/or defective tails (bent, folded, or coiled tails). Contrarily, at T 48 of storage, the intact acrosome percent was significantly lower (p<0.05) in control and caffeinetreated groups, compared to those sup plemented with melatonin or omega 3. In the meantime, the control group recorded the lowest (p<0.05) percent of intact sperm cell membrane, as determined by HOS test, compared to all supplemented groups ( Table 2).

Effects on oxidative status during 48 h of liquid-chilled storage
The levels of TAC in preservation medium decreased signifi cantly (p<0.05) in both control and caffeinetreated semen over time of storage, reaching the lowest (p<0.05) values at T48 compared to other supplemented specimens (Table 3). Contrariwise, MDA concentration increased (p<0.05) in the control group and reached the highest (p<0.05) level at T 48 compared to all treated groups (Table 3). No significant dif ference was observed in reduction of RRT among control and treated specimens over time of storage (Table 3). On the other hand, enzymatic activities of ALT, AST, and ALP in preser vation medium increased significantly (p<0.05) in the control group over the 48 h preservation period recording the highest (p<0.05) activities at T 48 compared to all treated specimens ( Table 3).

Effects on post-thaw physical and kinematic sperm properties
The CASAderived assessment revealed that the omega 3 treated specimens recorded the highest (p<0.05) postthaw progressive motility (%), whereas the control group recorded the lowest (p<0.05) value ( Figure 1). Meanwhile, the control group showed the lowest (p<0.05) percentages of sperm viabil ity and normal spermatozoa compared to each of caffeine, melatonin and omega 3 treated groups (Figure 1). Even though no significant difference was observed in postthaw primary abnormalities among control and treated groups, the percent of secondary sperm abnormalities was significantly higher (p<0.05) in the control group compared to all other treated specimens (Figure 1). Similarly, the results showed that the percent of intact acrosome was significantly lower (p<0.05) in the control group compared to all other treat ments ( Figure 1). Concurrently, both control and caffeine supplemented groups exhibited the lowest (p<0.05) percentages of integrated cell membrane compared to melatonin and omega 3 supplemented groups (Figure 1). The results of postthaw sperm kinematics showed that inclusion of the three additives in cryopreservation medium affected (p<0.05) all sperm motion and velocity patterns ex cept for motion indices of LIN (%) and STR (%) (Figure 2).
In this regard, the percent of postthaw progressive motility, particularly those of classA and B, was higher (p<0.05) in both melatonin and omega 3supplemented groups compared to those of control and caffeinesupplemented specimens. Furthermore, the melatonin supplemented group exhibited the highest (p<0.05) percent of nonprogressive motility and VAP (μm/s), while recording the lowest (p<0.05) percent of immotile spermatozoa compared to all other groups ( Figure  2). On the other side, values of ALH (μm) were significantly higher (p<0.05) in the omega3 group (2.7±0.2 μm) than those observed in control (1.8±0.3 μm) and caffeinesupplemented group (1.6±0.2 μm). In addition, the results demonstrated an improvement (p<0.05) in VCL (μm/s) and VSL (μm/s) in all supplemented specimens compared to that of control (Fig  ure 2).

DISCUSSION
Exposing spermatozoa to coldshock through chilled storage or cryopreservation alters their cell membrane structure and function due to spontaneous lipid peroxidase reaction (LPO) and increased MDA production. These alterations comprise redistribution of membrane bound phospholipids and pro teins as well as membrane permeability and ion exchange [20], which decrease sperm viability and fertility [21] and, even tually, cause sperm death [22]. Ram spermatozoa, having high   concentration of PUFAs in their cell membranes, are excep tionally vulnerable to such stress with subsequent reduced sperm motility and loss of sperm functional integrity during chilled or cryo preservation [2,5].
The present results clarified that caffeine supplementation drastically reduced sperm motility and increased secondary morphological abnormalities over chilledpreservation time compared to both melatonin and omega3. This was com bined with decreased TAC and elevated LPO and enzymatic activities in caffeinetreated specimens compared to the other treated groups. Furthermore, although incorporating caffeine into cryopreservation medium increased VCL, VSL, VAP, LIN, and WOB compared to melatonin and omega 3treated specimens, it negatively affected integrity of sperm cell mem branes. This is in consonance with previous results in rams [23]. Contrariwise, improved sperm acrosome integrity has been reported after caffeine inclusion in sperm diluent of buffalos [24] and camels [25].
Caffeine (1,3,7trimethylxanthine) is a natural stimulant belonging to the methylxanthine class, and has been utilized as a supplement in sperm capacitation medium to enhance progressive motility in IVF schemes. Caffeine inhibits cyclic nucleotide phosphodiesterase resulting in increased intracel lular cyclic adenosine monophosphate. The later acts directly on sperm plasma membrane channels to increase intracellu lar calcium ions in sperm flagella and induces immediate sperm hyperactivation [11]. Hyperactivation is commonly observed in spermatozoa undergoing capacitation [26], which may explain the increased levels of altered acrosome/cell mem branes observed in caffeinetreated specimens, particularly those subjected to liquidchilled storage, in the current inves tigation. Furthermore, sperm hyperactivation implies the high energy state of spermatozoa. This was reflected in the increased sperm kinematic criteria observed in caffeinetreated specimens in the present results. Such increase in sperm kinematics could be attributed to ability of caffeine to activate glycogen phosphorylase and consequent glycogen break down into simple sugars [27], available for consumption by spermatozoa to cope with stimulated motility.
On the other side, our results clarified the antioxidant ca pabilities of melatonin supplementation on counteracting detrimental effects of LPO reaction on chilled and cryopre served spermatozoa. In addition to its multiple actions on different physiological processes, melatonin and its metabo lites are considered powerful antioxidants due to their ability to scavenge excessive ROS and, thus, protect spermatozoa [28]. Furthermore, melatonin has the potency to improve mitochondrial health state and functions via modulating glu tathione activity thereby improving IVF outcomes [10].
The results also demonstrated the protective effects of omega3 PUFAs on maintaining sperm physical properties and functional integrity during both liquidchilled and cryo storage. These results are in accordance with those reported previously in rams [14]. Omega3 comprises a group of es sential long chain PUFAs that influence biosynthetic pathways involved in regulation of animals' various functions includ ing reproduc¬tion. The EPA, DHA, and ALA are considered the 3 major n3 PUFAs that target reproductive cells and alter reproductive function and fertility [29]. These n3 PUFAs act directly on sperm membrane lipid composition to promote creation of microdomains with different flexibility, fluidity, fusogenicity, and permeability characteristics, hence, they were regarded as the major determinants of sperm mobility characteristics, cold sensitivity, viability and membrane in tegrity [30]. Moreover, they provide spermatozoa with energy and regulate membrane proteins thereby maintaining sperm viability during exposure to cold stress [31].
In summary, our results accentuated potency of both mela tonin and omega3 PUFAs when incorporated into chilled and cryopreservation medium on ameliorating the deleterious effects of oxidative stress and, consequently, maintaining sperm physical and kinematic properties compared to caffeine sup plementation. Further studies are still needed to declare the effects of inclusion of these additives in sperm preservation medium on AI and IVF outcomes.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.