Influences of Monosaccharides and Disaccharides Supplementations in Tris Media on the Motility Patterns of Fresh and Chilled Small Ruminant Spermatozoa

Document Type : Research Article

Author

• Mazen Alomar

Division of Animal Production, Department of Agriculture, Atomic Energy Commission, Damascus, Syria.

10.22067/ijvst.2023.82141.1247

Abstract

In this study, the effects of monosaccharides, including glucose and fructose, and disaccharides, namely sucrose and trehalose, in eight Tris media on the motility patterns of small ruminants spermatozoa were investigated. Fresh and chilled semen samples from five Awassi rams and five Shami bucks were diluted in TBM and TEY containing 50 mM of the four different sugar types. The characteristics of spermatozoa motility were analyzed using a computer-assisted sperm analyzer (CASA). Fresh ram spermatozoa incubated in a TBM-fructose medium had the highest CASA values with no differences between the motility values generated from the fructose- and glucose-supplemented media. Trehalose reduced the values of velocity parameters, including VAP, VCL, and VSL for fresh ram sperm. Sucrose was the most influential sugar in raising the values of motility parameters MOT%, PMOT%, VAP, VCL, and VSL for fresh bucks spermatozoa, while trehalose generally had an important positive effect on chilled buck sperms. No significant differences (p > 0.05) were recorded for sperm trajectory parameters where the values of STR% and LIN% for the two ruminant species and the two spermatozoa types did not significantly differ between the eight media. It was concluded that during the first hours of in vitro incubation and based on the incubation temperature, the velocity parameters of small ruminant spermatozoa were the most affected CASA characteristics by monosaccharides and disaccharides supplementations in Tris semen media.

Keywords

• Spermatozoa
• Motility
• CASA
• Glucose
• Fructose
• Sucrose
• Trehalose

Main Subjects

• Reproductive

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Abbreviations

AECS :Atomic Energy Commission of Syria

CASA: computer-assisted sperm analyzer

GLM: general linear model procedure

IVF: in vitro fertilization

LIN%: linearity percent

MDA: malondialdehyde

MOT% :motility percent

PMOT%: percent of sperm showing progressive motility

STR%: straightness percent

TBM: Tris-based media

Introduction

It is well known that semen preservation media are needed to maintain spermatozoa viability and motility of the different animal species [ 1 ]. However, to sustain the motility status, spermatozoa require proper nutritional agents in the media. In this regard, sugar is one of the main constituents in semen media which can be easily metabolized into energy [ 2 , 3 ]. Motility has been considered one of the most important indicators of sugar utilization by sperm because sugars provide the external energy source necessary for maintaining the motility status [ 2 , 4 ]. It must be noted that when the environment does not provide any external energy source for the semen, the spermatozoa could use sugars in two ways: 1) obtaining energy by the Krebs cycle [ 5 ] and 2) storing sugars in the form of glycogen which presents a middle- to long-term energy reserve that could maintain motility [ 4 ].

Generally, sugars are divided into three major groups: monosaccharide, disaccharide, and trisaccharide. Monosaccharides are the main energy source for mammalian sperm [ 3 ], and fructose and glucose are the most important members of this sugar group. Fructose is an essential source of spermatozoa energy as it is metabolized and converted to pyruvate and lactate to support both sperm motility and viability [ 6 ]. In certain conditions, fructose acts as an extracellular cryoprotectant agent to protect the sperm membrane from toxicity during storage [ 7 ]. Glucose is also essential for energy utilization by spermatozoa [ 8 ]. In humans, glucose is required to sustain an optimal ATP concentration and to support optimum sperm motility [ 9 ]. Moreover, according to previous studies, glucose can support significant levels of hyperactivated motility and at the same time can be substituted by fructose.

Sucrose and trehalose are two disaccharides with the same molecular formula (C₁₂H₂₂O₁₁) but with different geometrical structures. Sucrose is composed of two monosaccharides, including glucose and fructose, while trehalose consists of two molecules of glucose. Sucrose is produced naturally in plants, from which table sugar is refined, while some bacteria, fungi, plants, and invertebrate animals synthesize trehalose as a source of energy. Sperm quality parameters of chilled and cryopreserved semen were shown to improve by using trehalose. The protective effects of this sugar significantly enhanced the freezability of buck and ram spermatozoa [ 10 , 11 , 12 ]. However, when trehalose was added in high concentration in a culture medium, sperm movement was hampered [ 13 ].

Sugar consumption by spermatozoa depends on both sugar type and sugar concentration. Matos-Brito et al. [ 14 ] showed that when extenders were used with appropriate concentrations of carbohydrates, goat sperm remained viable regardless of the initial concentration of fructose in goat seminal plasma. Moreover, Salamon and Ritar [ 15 ] noted that glucose and fructose addition to Tris buffer extenders resulted in higher post-thaw motility. Despite the importance of all the previously mentioned studies, the direct actions of sugars on spermatozoa are still little understood. In addition, to achieve the most efficacious use of fresh and chilled small ruminant semen, it is important to study the influence of diverse sugars on the motility status of these two spermatozoa types preserved in different semen media. Thus, the main objective of the present study was to assess the effects of using monosaccharides and disaccharides in Tris semen preservation media on the motility patterns of small ruminants spermatozoa, including fresh and chilled rams and bucks spermatozoa, during the first hours of in vitro incubation.

Results

Table 1 shows CASA motility values of fresh spermatozoa from rams incubated in TBM media supplemented with 50 mM of glucose, fructose, sucrose, and trehalose at 37°C for 60 min. The highest values of MOT% and PMOT%, as well as the velocity parameters VAP, VSL, and VCL were recorded when spermatozoa were incubated in the TBM solution containing fructose without recording any significant differences for MOT% and PMOT% between the four media. The addition of trehalose to the TBM solution led to a significant (p < 0.05) decrease in the values of VAP, VSL, and VCL in comparison with the spermatozoa incubated in TBM-sucrose, TBM-glucose, and TBM-fructose media. No significant differences (p > 0.05) were recorded between the four media containing monosaccharides and disaccharides for the trajectory parameters STR% and LIN%.

Table 1.CASA sperm motion characteristics of fresh spermatozoa from rams incubated in Tris based medium (TBM) supplemented with 50 mM of glucose, fructose, sucrose and trehalose at 37°C for 60 minutes. Mean (± Sd) of CASA parameters: average path velocity (VAP), straight line velocity (VCL), curvilinear velocity (VCL), per- cent straightness (STR %), Percent linearity (LIN %) and the distribution percentage of motility subpopulation.

CASA Parameters	Glucose	Fructose	Sucrose	Trehalose
Motlity %	89.44 ± 2.92a	93.5 ± 1.88a	91.89 ± 2.26a	89.55 ± 0.73a
Progressive motility %	20.44 ± 1.33a	22.56 ± 2.88a	19.44 ± 1.42a	18.66 ± 7.3a
VAP (µm/s)	115.11 ± 4.01ab	120.6 ± 11.9 a	114.89 ± 5.98ab	109.33 ± 8.26b
VSL (µm/s)	74.89 ± 4.04ab	78.44 ± 9.25a	69.67 ± 3.73ab	67.89 ± 4.26b
VCL (µm/s)	226.44 ± 7.32ab	234.6 ± 11.4 a	221.44 ± 7.52ab	210 ± 5.35b
STR %	61.33 ± 2.65a	59.88 ± 3.01a	58.22 ± 1.79a	60.44 ± 1.51a
LIN %	33.22 ± 2.04a	32.56 ± 2.3a	30.33 ± 1.80a	33 ± 1a
Motility subpopulations
Static %	11.22 ± 2.89a	7.89 ± 3.05a	8.89 ± 1.85a	11.31 ± 1.73a
Slow %	5.44 ± 2.89a	4.55 ± 1.51a	5.33 ± 1.69a	10.48 ± 3.28b
Medium %	23.77 ± 3.53a	22.44 ± 2.52a	26.22 ± 2.92a	24.64 ± 1.92a
Rapid %	59.55 ± 1.36ab	65.11 ± 3.05a	59.56 ± 1.37ab	53.55 ± 1.37b
The means (± Sd) with different letters (a-b) within columns significantly differ at p < 0.05.

Table 2 shows CASA motility values of fresh spermatozoa from bucks incubated in TBM media supplemented with 50 mM of glucose, fructose, sucrose, and trehalose at 37°C for 60 min. The effect of sucrose was evident by increasing the values of MOT%, PMOT%, VAP, VSL, and VCL parameters in comparison with the other three sugar types. The clearest difference in motility values was between the medium containing sucrose and the base solution containing trehaloseDiscussion (29.11 vs. 21.11 for PMOT%, and 130.11 vs. 107.66 for VAP, respectively), while no significant differences were recorded between the four TBM media for STR% and LIN%. Moreover, sucrose was also able to significantly raise (p < 0.05) the percentage of rapid sperm subpopulation compared to the other three media.

Table 2.CASA sperm motion characteristics of fresh spermatozoa from bucks incubated in Tris based medium (TBM) supplemented with 50 mM of glucose, fructose, sucrose and trehalose at 37 °C for 60 minutes. Mean (± Sd) of CASA parameters: average path velocity (VAP), straight line velocity (VCL), curvilinear velocity (VCL), per- cent straightness (STR %), Percent linearity (LIN %) and the distribution percentage of motility subpopulation.

CASA Parameters	Glucose	Fructose	Sucrose	Trehalose
Motlity %	87.67 ± 4.24 ab	89.6 ± 2.1ab	92.77 ± 2.58 a	85.11± 3.98b
Progressive motility %	20.77 ± 1.86b	25 ± 1.73ab	29.11 ± 1.61a	21.11 ± 2.89b
VAP (µm/s)	102.33 ± 5.34 b	114.4 ± 4.21a	130.11± 8.79 c	107.66 ± 7.26b
VSL (µm/s)	65.89 ± 2.26 b	75 ± 4.39a	92.33 ± 7.78 c	71.33 ± 6.32 a
VCL (µm/s)	209 ± 5.12 b	215.6 ± 8.59a	228.11± 12.25a	211.22 ± 8.3ab
STR %	58.66 ± 2.45a	60.00 ±1.53a	63.33 ± 1.23a	60.77 ± 2. 6 a
LIN %	31.66 ± 1a	34.11± 0.93 a	36.55 ± 2.06 a	34.77 ± 1.92 a
Motility subpopulations
Static %	12.01 ± 3.46a	11.66 ± 3.84 a	7.44 ± 2.13 b	14.77 ±3.11 a
Slow %	5.66 ± 1.72a	4.33 ± 0.77 a	3.88 ± 0.38a	3.11 ± 0.86 a
Medium %	14.01 ± 2.11a	11.11 ± 0.96 a	9 ± 1.61b	12.88 ± 3.57a
Rapid %	68.33 ± 2.31a	73.11 ± 3.07 a	79.77 ± 2.94b	69.33 ± 4.35a
The means (± Sd) with different letters (a-b) within columns significantly differ at p < 0.05.

Table 3 shows CASA motility values of chilled spermatozoa from rams incubated in TEY media supplemented with 50 mM of glucose, fructose, sucrose, and trehalose at 5°C for 180 min. No differences were noted for none of the CASA motility characteristics between the chilled ram spermatozoa samples treated with different sugar types. Table 4 shows CASA motility values of chilled spermatozoa from bucks incubated in TEY media supplemented with 50 mM of glucose, fructose, sucrose, and trehalose at 5°C for 180 min. No significant differences (p > 0.05) were recorded for sperm incubated within TEY media containing the four types of supplemented sugars for both MOT% and PMOT% parameters. An increase in VAP, VCL, and VSL values was observed for the spermatozoa incubated in TEY medium containing trehalose, while there were no significant differences (p > 0.05) in the values of STR% and LIN % between the four different TEY media. Moreover, compared to the incubated spermatozoa in the TEY-fructose medium, a rise in the percentage of the rapid subpopulation category was caused by trehalose supplementation, and a decrease in the percentage of static sperm was recorded in this medium.

Table 3.CASA sperm motion characteristics of chilled spermatozoa from rams incubated in Tris egg-yolk medium (TEY) supplemented with 50 mM of glucose, fructose, sucrose and trehalose at 5 °C for 180 minutes. Mean (± Sd) of CASA parameters: average path velocity (VAP), straight line velocity (VCL), curvilinear velocity (VCL), per- cent straightness (STR %), Percent linearity (LIN %) and the distribution percentage of motility subpopulation.

CASA Parameters	Glucose	Fructose	Sucrose	Trehalose
Motlity %	91.55 ± 2.3a	91.11 ± 1.96a	92.67 ± 2.5a	91.56 ± 1.94a
Progressive motility %	16.55 ± 2.45a	16.44 ± 2.24a	16.11 ± 3.01a	15.11 ± 2.52 a
VAP (µm/s)	105.55 ± 7.23a	107.3 ± 6.42a	100.77 ± 4.52 a	99.11± 3.88 a
VSL (µm/s)	67 ± 5.12a	67.33 ± 4.58a	65.89 ± 4.25a	64.89 ± 4.98a
VCL (µm/s)	205.44 ± 8.35a	207.11 ± 6a	203.55 ± 4.36 a	199 ± 8.1 a
STR %	58.88 ± 1.76 a	59.33 ± 1.73a	58.77 ± 1.72a	60 ± 2.4 a
LIN %	31.33 ± 1a	31.78 ± 1.30a	31.55 ± 1.9a	32 ± 2.18 a
Motility subpopulations
Static %	9.77 ± 1.71a	9.11 ± 1.39a	8 ± 1.89a	9.78 ± 2.58a
Slow %	6.22 ± 1.84a	7.55 ± 2.04a	6.88 ± 1.66a	8.22 ± 2.71a
Medium %	29.44 ± 2.93a	27.77 ± 2.15a	29.88 ± 1.40a	29.66 ± 1.24 a
Rapid %	54.55 ± 4.72a	55.55 ± 2.77a	55 ± 1.59a	52.11 ± 2.57a

Table 4.CASA sperm motion characteristics of chilled spermatozoa from bucks incubated in Tris egg-yolk medium (TEY) supplemented with 50 mM of glucose, fructose, sucrose and trehalose at 5 °C for 180 minutes. Mean (± Sd) of CASA parameters: average path velocity (VAP), straight line velocity (VCL), curvilinear velocity (VCL), per- cent straightness (STR %), Percent linearity (LIN %) and the distribution percentage of motility subpopulation.

CASA Parameters	Glucose	Fructose	Sucrose	Trehalose
Motlity %	90.77 ± 3.15a	90.44 ± 2a	89.77 ± 1.56 a	93.22 ± 2.28a
Progressive motility %	20.11± 2.82a	20 ± 2.7 a	19.44 ±1.23 a	22.11 ± 2.57a
VAP (µm/s)	86.56 ±9.14b	87.8± 5.31ab	92.22 ± 4.9 ab	96.77 ± 5.26 a
VSL (µm/s)	60.22 ± 6.30a	60.44 ± 3.17a	62.44 ± 2.9 ab	65.89 ± 5.13 b
VCL (µm/s)	184.66 ±10.16a	186.4 ± 9.72a	189.22 ± 7.07a	197.22 ± 4.57 b
STR %	61.66 ± 1.5 a	61.11± 2.93a	60.44 ± 0.72a	61.78 ± 2.28a
LIN %	32.78 ± 2.39 a	32.44 ± 1. 9a	32.22 ±1.09a	32.77 ± 1.72a
Motility subpopulations
Static %	10.78 ± 2.38 a	10.33 ± 0.96 a	11.11 ± 1.36 a	8 ± 0.91b
Slow %	8.77 ± 1.94 a	8.44 ± 1.19 a	8.55 ± 1.59a	6.78 ±1.03a
Medium %	21.78 ± 1.93a	22.55 ± 1.72 a	21 ± 2.73a	20.88 ±3.29a
Rapid %	58.66 ± 2.80ab	58.22 ± 2.10 a	59.44 ± 2.66ab	64.33 ±3.70b
The means (± Sd) with different letters (a-b) within columns significantly differ at p < 0.05.

Discussion

Our present study is the first investigation that simultaneously shows the direct effects of monosaccharides and disaccharides on the spermatozoa motility pattern of two small ruminant species, including sheep and goat, and two spermatozoa types, including fresh and chilled samples. It must be stressed that motility is the main function of sperm cells and the main aim of energy obtainment. In this respect, sugar can be easily changed into energy, and the use of sugars, such as fructose, glucose, sucrose, and trehalose in semen media could increase sperm motility. It is well known that spermatozoa is a strict, glycolytic cell. Furthermore, the predominant metabolic pathways through which spermatozoa produce ATP, necessary for sperm motility, are mitochondrial oxidative phosphorylation and glycolysis [ 9 ]. In sperm cells, sugar, and especially glucose, is the main substrate for glycolysis, where it is metabolized to pyruvate and/or lactate to obtain cellular energy in the form of ATP. Generally, the effects of sugars may largely vary between species due to the differences in the chemical and physical composition of the sperm [ 16 , 8 ]. Moreover, the differences in spermatozoa motility status may be due to several factors, including individual variation [ 17 , 18 ], storage temperature [ 19 ], type of media [ 20 ], and also the differences in motility estimation methods (subjective or objective).

As sperm cells are dependent on their storage medium, different semen media, such as TBM and TEY, were developed to be used in assisted reproductive technologies. In this respect, sugar concentration is one of the most important points that should always be carefully considered during the preparation of any semen media, especially TBM and TEY. In the present study, a concentration of 50 mM for each sugar type was adopted in TBM and TEY media, as one of the concentrations usually used in Tris media [ 21 ]. Better maintenance of canine spermatozoa motility was noted when increased amounts of sugars were added to the semen extender. In this respect, the supplementation of TEY extender with 70 mM of sugars had notable beneficial effects on chilled canine spermatozoa compared to 10 mM, and this concentration resulted in significantly higher values for the percent motility and VAP parameter over the experiment period [ 22 ]. Moreover, compared to the freshly pooled canine semen, the mean values of VAP, VSL, and VCL increased significantly, suggesting that sugars activate sperm velocity [ 22 ]. However, in the present case, any higher concentrations over the 50 mM level of monosaccharides or disaccharides may cause osmolarity to become too high and in such a situation, substantial osmotic damage could be produced. It is well known that sperm cells are sensitive to osmotic stress [ 21 , 23 ]. However, spermatozoa can tolerate a moderate range of osmolarities without a reduction in fertility. In this study, the osmolarity of the eight Tris media was within the range of physiological values (300-330 mOsm/Kg), which is the physiological osmolarity of most physiological fluids. Therefore, the osmolarity levels in our Tris media are not expected to have any significant effect on the motility status of rams and bucks spermatozoa.

The effects of monosaccharides on the metabolism of freshly ejaculated spermatozoa are very important for motility status. In dogs, sperm metabolize fructose and glucose in separate pathways resulting in separate systems of energy management indicated by different motility patterns and different roles in glycogen metabolism [ 2 , 4 ]. The activating role of fructose and glucose on dog sperm was initiated by the intense and rapid increase in the tyrosine phosphorylation of some specific proteins [ 2 ]. Sperm functions could be modulated and modified immediately after ejaculation. However, further research in protein phosphorylation in the spermatozoa of small ruminant species treated by different sugar types must be conducted in the future. On the other hand, is it possible to make a preference for a specific type of monosaccharides to be used in semen solutions? In general, the sperm of many mammals use glucose in preference to fructose when both substrates are available [ 24 , 25 ]. Rogers and Perreault [ 26 ] observed that glucose supported better progressive human sperm motility than fructose, while Williams and Ford [ 9 ] found both sugars to be equally effective. In contrast, fructose has been found beneficial for frozen-thawed sperm in bovine [ 27 ] and ram [ 28 ]. It must be noted that in goat seminal plasma, fructose was the primary substrate for glycolysis [ 29 ] and the end product of the glycolysis pathway produces more ATP, necessary to support sperm motility. However, the combination of glucose and fructose as a supplement in the TALP medium improved the progressive motility of boar sperms [ 30 ]. In our study and during the first hours of in vitro incubation, we did not notice any clear differences between glucose and fructose, especially for the fresh and chilled ram spermatozoa. In agreement with these results, the survival of unfrozen ram sperms did not improve when ten different sugars were added [ 20 ]. Moreover, no effect was noted for sugar type (i.e., glucose, fructose, sucrose, lactose, and trehalose) on the motility of the post-thaw ram spermatozoa [ 31 ]. It must be pointed out that the monosaccharide metabolization pathways in mammalian spermatozoa depend on several factors, such as the medium composition, energy consumption rhythm, and most importantly the studied species where the sperm come from [ 3 ].

Diverse sperm types, semen media, and sugar groups used for supplementation could result in different motility patterns. In this study and unlike the motility status in the case of fresh spermatozoa types, trehalose had positive effects on CASA velocity parameters and the rapid subpopulation category of chilled goat spermatozoa. In contrast to our findings concerning chilled ram spermatozoa, the results of Zhao et al. [ 12 ] indicated that the addition of trehalose to Tris diluent improved the quality of long-preserved ram semen under low-temperature conditions. According to the previous authors, trehalose addition to Tris-fructose egg yolk medium at a concentration of less than 20 mM did not directly increase the progressive motility of sperm but significantly raised the integrity of the preserved sperm acrosome. In humans, superior post-thaw sperm parameters were observed by using 50 mM trehalose over sucrose and other trehalose concentrations [ 32 ]. It must be noted that trehalose exerts an indirect antioxidant effect by augmenting the level of glutathione and reducing lipid peroxide [ 33 ]. Chhillar et al. [ 34 ] documented that trehalose decreased H2O2 and MDA in frozen-thawed bull semen to the levels of fresh semen, while Badr et al. [ 35 ] reported similar results in buffalo. However, the antioxidant potential of trehalose on the spermatozoa of small ruminants and its relation to sperm motility pattern needs further research.

In the present study, we focused on fresh and chilled spermatozoa types. It must be pointed out that these two types could be used in different assisted reproductive technologies and they present a very important option for using cryopreserved ones. As the cryoprotective effects of monosaccharides and disaccharides on spermatozoa were well documented in the literature [ 36 , 37 ], our present study was more focused on the motility aspect. However, the main result demonstrated that regardless of temperature, both monosaccharides and disaccharides could support the motility of bucks and rams spermatozoa during the first three hours of in vitro incubation in the Tris media.

Finally, two major limitations could be noted for the current study. The first is that sperm CASA motility parameters may not accurately predict the in vivo fertility results or IVF outcome. Therefore, other assessments of semen quality, including sperm viability, membrane, and acrosome integrity are needed. The second limitation is that motility status in this study was assessed during the first hours of in vitro incubation. In fact, we were interested in these particular incubation time points for both fresh and chilled spermatozoa samples because they represent the time window by which spermatozoa of such types could be directly used in different assisted reproduction technologies, especially AI. However, during the first three hours of in vitro incubation, our initial data clearly showed that the four sugars only affected the velocity, and not the spermatozoa trajectory of the small ruminants.

Conclusion

Taken together and by using CASA technology, this research showed the direct effects of monosaccharides and disaccharides supplementations in different Tris media on the spermatozoa motility patterns of small ruminants. It was clear that the sperm motility parameters of both rams and bucks were affected by monosaccharides and disaccharides based on the incubation temperature. Moreover, CASA velocity parameters were the most affected motility characteristics by sugars, while no differences were recorded for the sperm trajectory parameters. Such data could assist in the selection of the most appropriate sugar compatible with the temperature of keeping small ruminant spermatozoa within the semen media to achieve the best motility status. However, further studies are needed to test the in vivo and in vitro fertilization of small ruminants' spermatozoa incubated in different media supplemented with different sugars.

Materials and Methods

Animals and ethical approval

This study was carried out at Der-Al-Hajar Animal Production Research Station, 33 km southeast of Damascus. Semen was obtained from five adult Awassi rams and five adult Shami bucks (two local Syrian small ruminant species). The animals aged 3-4 years, and were fed a diet based on concentrate, wheat straw, and barley, with water available ad libilum. It must be noted that the experiments for this study were approved by the Local Scientific and Ethical Committee of the AECS, Damascus, Syria (permit number 36-Z/M4 - 2019).

Media preparation, semen collection, and experimental design

All chemicals were purchased from Roth (Carl Roth Gmbh-Karlsruhe-Germany). Eight Tris media were prepared in the present study, including four TBM and four TEY. TBM prepared as a 300 mOsmol/Kg solution contained 200 mM tris (hydroxymethyl) aminomethane, 64.7 mM citric acid monohydrate, and 50 mM of each sugar type (glucose, fructose, sucrose, and trehalose), which were separately added to each base solution. For the TEY media, 20% of egg yolk was added to the four tris-based solutions with conserving the same previous concentrations of tris (200 mM), citric acid (64.7 mM), and the four sugar types (50 mM for each) in each TEY medium. It must be noted that the eight media were always held constant at pH 7.

In this study, a total of 60 ejaculates were collected from ten experimental animals (30 ejaculates from rams and 30 from bucks). Semen samples were collected using an electro-ejaculator (Minitube-Electro Ejaculator, Tiefenbach, Germany). Upon collection, semen specimens were immediately evaluated for general appearance and volume. For each animal and after semen collection, spermatozoa concentration was estimated using a haematocytometer (Neubauer Improved Marienfeld, Germany). An initial analysis of sperm motility was performed using the CASA system (Hamilton Thorne Biosciences, Version 12 CEROS, Beverly, USA). Sperm samples with a motility score ≥ 75% of motile spermatozoa and a concentration of ≥ 1×10⁹ spermatozoa/ml were utilized. All ejaculations with no or poor motility status were immediately excluded before conducting the analyses. The collected ejaculates from each species were mixed in each replicate to isolate the individual effect of males. The fresh spermatozoa samples (25×10⁶/ml) of both bucks or rams were incubated in TBM containing the four sugar types at 37°C in a water bath for 60 min, while the chilled spermatozoa samples (25×10⁶/ml) of bucks or rams were incubated in TEY media containing the four sugar types at 5°C in the refrigerator for 3 h. Each experiment for each animal species and each semen media was repeated three times.

Motility assessment

The motility characteristics of the spermatozoa were assessed by CASA, using the Hamilton-Thorne motility analyzer (Hamilton Thorne Biosciences, Version 12 CEROS, Beverly, USA). For each sperm sample, three fields were selected, counted randomly, and assessed to generate data from at least 200-250 sperm/samples. The CASA characteristics included in the analysis were the MOT%, VCL (µm/s), VAP (µm/s), VSL (µm/s), LIN%, STR%, and PMOT% (VAP ≥ 75 µm/s and STR ≥ 80 %; HTM-CEROS; installation getting started guide version 12 CEROS). Spermatozoa subpopulations were defined in four categories by CASA as Rapid (4): fraction of all cells moving with VAP = 25 µm/s; Medium (3): fraction of all cells moving with 5 µm/s < VAP ≤ 25 µm/s; Slow (2): fraction of all cells moving with VAP < (5 µm/s or VSL < 11 µm/s; and Static (0-1): fraction of all cells not moving at all.

The Hamilton-Thorne motility analyzer settings used for goat spermatozoa were negative phase contrast optics at a recording rate of 60 frame/s, temperature of analysis 37°C, light adjustment 90-110, minimum cell size 5 pixels, non-motile head size 10 pixels, non-motile head intensity 80, low VAP cut off 20 µm/s, low VSL cut off 5 µm/s, static size limit 0.60/4.32 (min/max), and static intensity limit 0.20/1.92 (min/max). While the Hamilton-Thorne motility analyzer settings used for sheep spermatozoa were negative phase contrast optics at a recording rate of 60 frame/s, temperature of analysis 37°C, light adjustment 90-110, minimum cell size 5 pixels, non-motile head size 10 pixels, non-motile head intensity 80, low VAP cut off 21.9 µm/s, low VSL cut off 6 µm/s, static size limit 0.60/8 (min/max), and static intensity limit 0.25/1.50 (min/max).

Statistical analysis

Statistical analysis was conducted using the Minitab program (Minitab Coventry, United Kingdom, Version 13.31, 2000). Motility data were subjected to a factorial analysis of variance (ANOVA) for the four sugar types by GLM, followed by multiple pairwise comparisons using the Tukey posthoc test. The threshold of signification was set at p < 0.05.

Acknowledgements

The author would like to thank the Atomic Energy Commission of Syria for their support while conducting the experiments.

Competing Interests

The author declare that there is no conflict of interest.

Abbreviations

TEY: Tris-egg yolk

VAP: average path velocity VCL:curvilinear velocity

VSL: straight line velocity

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