Seasonal Variations in Semen Parameters of Zulu Rams Preserved at 10°C for 72 H During Breeding and Non- Breeding Season

Corresponding Author: Tshimangadzo Lucky Nedambale Department of Animal Sciences, Tshwane University of Technology, Staatsartillerie Rd, Pretoria West, Pretoria, 0183, South Africa Email: NedambaleTL@tut.ac.za Abstract: The study was conducted to evaluate the seasonal variations in semen parameters of Zulu rams preserved at 10°C for 72 h. The study site was the Agricultural Research Council (ARC) Irene, during the breeding season and the non-breeding season. Zulu rams (n = 6, age = 3 years and average weight = 35 kg ±2.29) were used in this study. Semen was collected with the aid of artificial vagina. Macroscopic (volume and pH), microscopic (sperm concentration, progressions, velocities and velocity ratios and morphology) semen parameters were evaluated immediately after semen collection. Data were analyzed using General Linear Model (GLM) in Minitab 17®. Semen volume was higher during breeding season (0.97±0.25 mL) than non-breeding season (0.72±0.5 mL). Total sperm motility was higher (92.01±1.40%) at 24h during breeding season than non-breeding season (88.69±1.40%), thereafter declined drastically in both seasons. However, non-breeding season yielded a high progressive motility (50.86±1.63%, 33.77±1.63% and 27.56±1.63%) after 24, 48 and 72 h than breeding season (21.32±1.63%, 11.89±1.63% and 10.29±1.63%). It was concluded that, semen parameters from Zulu rams does vary with seasons. Nevertheless, despite seasonal variations observed, Zulu rams’ semen quality is acceptable in both breeding and non-breeding season hence can breed throughout the year. Notwithstanding, studies involving fertility are recommended and the nutrition effect of lambing during dry period should be considered.


Introduction
Zulu sheep is a fat-tailed South African indigenous sheep along with BaPedi and Swazi breed, hardy and well adapting to harsh environmental conditions (Ramsay et al., 2000;Kunene et al., 2011). Zulu people brought this breed to the east coast of South Africa 200-400 AD ago hence named after Zulu tribe (Mavule et al., 2013;Du Toit, 2018). Despite their adaptability, Zulu sheep are endangered, with the breeding rams' numbers left not known with only 48 semen straws cryopreserved (FAO-DAD-IS, 2020) hence Cryo-gene bank is required urgently. Other studies on Zulu sheep revealed that they suffer from inbreeding depression (Kunene et al., 2009;Chella et al., 2017;Selepe et al., 2018) due to uncontrolled extensive rearing (Mavule et al., 2016).
Genetic improvement of endangered species and the control of diseases affecting their conservation programmes and production are vital (Bailey et al., 2000) and can be accomplished through Advanced Reproductive biotechnologies (ART) (Palacín et al., 2012). For example, using Artificial Insemination (AI), one ejaculate from a superior ram can impregnate multiple females depending on the dilution ratio, semen quality and sperm concentration (Paulenz et al., 2002), limiting physical contacts, spread of contagious diseases and introducing superior genotypes (Kershaw et al., 2005). Besides that, other ART such as semen cryopreservation facilitates long-term semen storage for proper transportation over distance locations thus enabling the use of superior sires even after death (Albiaty et al., 2016).
Alternatively, ram semen can and has been stored in a liquid form at different storage temperatures ranging from 4°C (Kasimanickam et al., 2007;, 5°C (Purdy, 2006;Purdy et al., 2010) and 15°C (Yániz et al., 2005;. However, when stored at 4°C and 5°C permanent Cryodamage has been observed due to the detrimental effects associated with cold shock (Gheller et al., 2018) targeting the plasma membrane (Bailey et al., 2000;Mortimer and Maxwell, 2004;Aitken and Nixon, 2013) hence failing to retain motility and lose fertility especially after 24 h (O'Hara et al., 2010). On other hand, (Yániz et al., 2010) revealed high metabolic activities when spermatozoa stored at 15°C resulting to the short lifespan not exceeding 24 h (Purdy et al., 2010).
No extensive studies on Zulu rams' semen parameters stored at 10°C and assessed by Sperm Class Analyser® (SCA) until this study so far. Likewise, no published study available addressing the preservation of Zulu sheep genetic resources using ART and preserved up to 72 h in both breeding and non-breeding season. Research published by (Chella et al., 2017) on Zulu sheep, covers the semen variation based on age, season in the fresh semen, with motility assessed subjectively. Therefore, it is vital to identify the seasonal variations in semen parameters from Zulu rams using objective semen evaluation methods such as Computer-Aided Sperm Analysis (CASA). This would favour the use of superior ram for successful cervical AI in Zulu sheep which will in turn improve in-vivo conservation program and yield comparable results to the sophisticated laparoscopic AI.

Study Ethics Approval
The Agricultural Research Council (ARC) and Tshwane University of Technology animal research ethics committee approved all study procedures with reference number: APIEC16/034 and AREC2018/03/004, respectively. The South African National Department of Agriculture, Land Reform and Rural Development (DALRRD) granted the section 20 of the Animal Disease Act (Act no. 35 of 1984).

Study Area
The study site was the Agricultural Research Council (ARC) Irene, small stock unit and Germplasm, Conservation and Reproduction Biotechnologies (GCRB) laboratory. The Agricultural Research Council area extends between 25º53'59.6'' South latitude and 28°12'51.6'' East longitudes. The ARC, Irene area situated in the Highveld at an altitude of 1525 m above sea level. According to (Grobbelaar et al., 2010), weather condition at ARC Irene ranges between hot days and cool nights in summer (17.5°C to 32°C) with moderate winter days and cold nights (1°C to 17°C).

Study Animals
Zulu rams (n = 6, age = 3 years, weight = 35 kg ±2.29) were used in this study. These rams were part of the Zulu sheep conservation program formed between the South African National Department of Agriculture, Land Reform and Rural Development (DALRRD) and Agricultural Research Council (ARC), Irene. All rams were raised at the ARC Irene. Initially (preliminary study), eighteen rams (average weight = ±35 kg 2.29 and age = 3 years) were selected from the base population, divided into the groups of 6 and kept at the camps with ±4 m 2 shelter to provide shades. Rams were taken cared for and fed balanced diet for maintenance as described by (NRC, 1985). Same diet was provided in both breeding and non-breeding season, consisting of free access to Eragrostis curvula hay and 400g of pellets per ram/day. Pellets were purchased from Meadow feeds with the nutrient composition of protein 15, moisture 12, fat 2.5, fiber 12, calcium 1.08, phosphorus 0.5, potassium 0.8 magnesium 0.08, sodium 1.1, chloride 1.7, sulphur 0.35%.
Famacha was conducted to detect internal parasite infections as described by (Ferreira et al., 2019), prior the commencement of semen collection. In case when internal parasites were suspected, Lintex-1 [Bayer South Africa, Registration number G447 (Act 36/1947)] was dosed orally, subjected to the Famacha score.

Semen Collection
Semen was collected with the aid of Artificial Vagina (AV) using estrogenized ewes as stimulus. These ewes (n = 4, aged = 3 years, average weight = ±35 kg) were estrous synchronized using Controlled Intravaginal Drug Released (CIDR) as described by (Ungerfeld and Rubianes, 2012) with few amendments. In brief, controlled intravaginal drug released was inserted into the vagina for seven days. On the 6th day, injection site was allocated and disinfected with the spirit to eliminate bacteria and 0.5 mL estrumate was injected subcutaneously.
During semen collection, rams were free from hunger and thirst, pain, injury and diseases, fear, distress and discomfort. Consequently, all expressed normal behaviour throughout the trial. However, in case where animal showing mild discomfort signs, was placed in the handling pen and habituated. Only experienced personnel were allowed to handle the animals.
Rams were given an adaptation period of ±10 days to human handling, using an ear massage every morning, precisely at a similar time every day (9 h00). After 5 days of habituation, all rams showed signs of comfort and calmness by allowing hand feeding and masticating during ear massage. Thereafter, a training to serve AV begun. All rams responded to AV training within three days and were advanced to the semen collection step. Three semen samples were collected during the preliminary study with the aid of artificial vagina and 2 days of resting period between collections was maintained. Semen was thereafter analyzed using Sperm Class Analysis (CASA). Only those produced semen with >90% total motility were considered for the experiment to limit individual variation. Those failed preliminary study tests were taken back to the base population. Hence, the final experimental sample size was six rams.

Semen Storage
Unless stated, all chemical products were purchased from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Steinem, Germany). The diluents were prepared using Sabax purified water (Adcock Ingram, South Africa PTY LTD). Commercial extender (Tris-based) was used for semen dilution and preservation. This was prepared on weekly basis and stored at 5°C (Hafez and Hafez, 2000;Salamon and Maxwell, 2000). Before use in the semen collection day, it was warmed up using warm plate adjusted to 37°C. The Tris-based extender was composited of Tris 1.211 g, citric acid 0.68 g, monohydrate glucose 0.5 g, sterile water 40 mL, egg yolk 10 mL and gentamycin sulphate 0.05 g and dissolved in 50 mL Falcon tube as described by (Salamon and Maxwell, 2000). For sperm washing solution, (dilution) egg yolk was not included. Trisbased extender was added to the semen at a ratio of 1:2 before storing at 10°C incubator [MCO-20 AIC Sanyo® CO2 incubator (Sanyo, Japan)]. Semen quality analysis was performed at fresh 0h and after 24, 48 and 72 h.

Semen Evaluation
Two hundred and eighty-eight ejaculates were collected during breeding season and another two hundred and eighty-eight ejaculates during non-breeding season. These gave the total of five hundred and seventysix ejaculates for the whole study. Late summer to autumn (May to July) was considered as a breeding season while late winter to spring (September to October) taken as a non-breeding season. Semen was evaluated as macroscopic (semen pH, volume), microscopic (sperm concentration, morphology and motility).

Macroscopic Semen Evaluation
Semen pH was measured using Oakton pH meter (Eutech Instruments Pte Ltd/Oakton Instrument, CyberScan pH 11/110). Semen volume was measured in milliliters (mL) using the graduation marks in the AV lining glass tube before dilution.

Microscopic Semen Evaluation
Spermatozoa concentration was evaluated with the use of grey Beckam spectrophotometer (Jenway 6310, Spectrophotometer, Bibby Scientific, England) as described by (Brito et al., 2016). Spermatozoa morphology and viability was examined using eosinnigrosin stain (Ondersterpoort Faculty of Veterinary Science' Pharmacy, South Africa). Stain was prewarmed in the warm plate to 37°C for about 5 min before mixed with semen. For smear, 20 μL of stain was pipetted to Eppendorf tube and mixed with 5 μL of semen. Five μL of mixed semen and stain was pipetted and placed near the labelled end of the microscope slide (76261 mm, Germany). The second microscope slide was held at 45° angle to the slide with the mixture of stain and semen. Thereafter was pulled gently without destroying spermatozoa as described by (Dolatpanah et al., 2008) across the slide. Smeared microscope slides were air dried for one day before examination. In the following day, 30 μm of oil immersion was pitted to the dried smeared slide and place under fluorescent microscope (Olympus BX 51TF, Japan), magnification (UPlanFLN 100/1.30) for morphology analysis. The spermatozoa morphology was recorded as abnormal mid-piece, loose head and tail, cytoplasmic droplets, bent and coiled tail (Bester et al., 2004). In each smeared slide, at least 200 cells were counted and percentage of mentioned defects was calculated and recorded on the Microsoft® Excel sheet.
Sperm motility was analyzed using Computer-Aided Sperm Analysis (CASA), Sperm Class Analyzer® system (SCA) 5.0 version (Microptic, Barcelona, Spain) at a magnification of 10 (Nikon, Japan). Sperm motility parameters evaluated were sperm progressions (progressive, non-progressive, total, slow, medium, rapid and static motility), sperm velocities (Curvilinear Velocity (VCL), Straight-Line Velocity (VSL) and average path Velocity (VSL)) and sperm velocity ratios (Linearity (LIN), Straightness (STR) and Wobble (WOB)). Careful precautions were taken to eliminate debris from egg yolk particles, known to affect CASA precisions (Holt et al., 2007). Five microliters of semen were diluted with one hundred microliters of extender (SWS) using Eppendorf tube. Thereafter, five microliters of the mixture were pipette and place in the warmed microscope slide (76261 mm, Germany) and gently enclosed with cover slip (2222 mm, Germany). Enclosed slide was placed over a warm plate (Omron) adjusted to 37°C, under the CASA camera at the magnification of 10 Ph1 BM. For each sample, three fields were randomly selected containing 100 to 200 spermatozoa and examined. Generated data was saved in the Microsoft Excel® sheet.

Statistical Analysis
Data was analyzed using General Linear Model (GLM) in Minitab 17® wherein the season (breeding and non-breeding), storage period and their interactions were fitted as independent variables. Means were compared using Fisher's LSD method and considered significantly when p<0.05.

Results
Data on the macroscopic semen analysis results are set out in Table 1. Semen volume (mL) during breeding season was higher (0.97±0.25 mL) than the non-breeding season (0.72±0.5 mL). Non-breeding season recorded high (6.83±0.54) semen pH than breeding season (6.54±0.98). Spermatozoa concentration did not differ (p>0.05) among seasons.

Discussion
Semen volume obtained in the current study fell within a normal range in both seasons. Ram semen volume normally ranges from 0.5 to 2.0 mL (Karagiannidis et al., 2000a;Hafez and Hafez, 2000) but highly affected by the collection frequency, season (Karagiannidis et al., 2000b), collection technique (Bopape et al., 2015), nutrition (Guan et al., 2014) and age (Chella et al., 2017). In temperate zones, photoperiod is the main factor defining the onset of breeding season (Pourseif and Moghaddam, 2012). Consequently, the evaluation of the fertilizing potential of a semen sample has been the utmost objective of semen analysis to estimate the fertility results of a future artificial insemination (Del Olmo et al., 2013). High semen volume obtained in the present study during breeding season was expected and in line with numerous studies (Barkawi et al., 2006;Talebi et al., 2009;Wang et al., 2015;Chella et al., 2017;. This is because, testicle volume shrink during non-breeding season and are highly affected by the heat stress (Hedia et al., 2019). Seminiferous tubules during breeding season occupies the majority of the testicular tissues (76.6%) and the spermatic layers (Barkawi et al., 2006;Kadili et al., 2019). Therefore, low semen volume obtained during non-breeding season is an indication of the positive correlation between semen volume and testicles size (Sarlós et al., 2013). Despite that, semen volume obtained in this study during non-breeding season fell within the normal range as reported previously (Karagiannidis et al., 2000a;Hafez and Hafez, 2000;Chella et al., 2017). Moreover, there was high differences among rams (data not shown) that was comparable to that reported by (Benia et al., 2018).
Semen pH is an exogenous factor influencing spermatozoa motility and its imbalances reduce spermatozoa motility, viability and capacitation (Hafez and Hafez, 2000). In this study, semen pH was higher (p<0.05) during non-breeding season than breeding season. According to (Chella et al., 2017) semen pH in Zulu rams' semen ideally ranges from 5.9 to 7.3. Moreover, (Al-Anazi et al., 2017), reported that semen pH in Naimi and Najdi rams was somewhat alkaline and lower during autumn than spring. Nevertheless, contradicted to (Talebi et al., 2009;Wang et al., 2015), where they found semen pH to not differ (p>0.05) throughout the year. However, this might due to the delays in semen processing after collection leading to acidic semen pH and the degradation of fructose by the sperm cells (Zamiri et al., 2010).
Ram sperm concentration ideally ranges from 3, 5109 to 6, 0109 sperm/ml (Hafez and Hafez, 2000). In the current study, spermatozoa concentration could not differ (p>0.05) among seasons (breeding and nonbreeding season). Benia et al. (2018) obtained similar results in Ouled-Djellal rams. However, other authors (Barkawi et al., 2006;Gündoğan, 2007;Azawi and Ismael, 2012;Golher et al., 2018;Hedia et al., 2019) reported huge seasonal variation on spermatozoa concentration, with breeding season recording higher concentration than non-breeding season with winter yielding lesser sperm concentration (Wang et al., 2015;Kadili et al., 2019). Noteworthy, the sperm concentration from the current study was higher than that reported by (Chella et al., 2017) in Zulu rams. Nevertheless, the effect pose by the environmental condition as well as the ability of each ram to respond to a particular environmental condition might explain these variations (Carvajal-Serna et al., 2019).
Semen was further evaluated for fresh spermatozoa motility in both breeding and non-breeding season. High total motility and rapid motility during breeding season than non-breeding season in the current study was expected in line to numerous studies (Gündoğan, 2007;Badi et al., 2018;Kadili et al., 2019). This is because semen quality is highly affected by testicular volume (Gündoğan, 2007) as mentioned above and scrotal circumference (Badi et al., 2018). Regardless of that, the effect of season in semen quality has been reported even in stallions (Janett et al., 2003), goats (Kumar et al., 2014) and Bulls (Perumal et al., 2017). Furthermore, although testosterone is not part of the current study, its high concentration during summer can explain these seasonal variations (Mallick et al., 2016). Ntemka et al. (2019) reported contradicting results where no significant different for sperm motility was declared between breeding and non-breeding season . Nevertheless, (Kulaksiz and Sen, 2019) reported high sperm motility and freezing-thawed survival during non-breeding season.
Breeding season recorded high non-progressive motility than non-breeding season. Whereas high progressive motility was attained during non-breeding than breeding season which were in line to (Kulaksiz and Sen, 2019). Previous studies reported seasonal variations of sperm quality numerous breeds ranging from United Arab Emirates crossbred rams (Ibrahim, 1997), Suffolk and Ile-de-France rams (Mandiki et al., 1998), indigenous breeds of Greece (Karagiannidis et al., 2000b) and Naimi and Najdi rams of Saudi Arabia (Al-Anazi et al., 2017). Therefore, this high progressive motility obtained during non-breeding indicate that Zulu rams produce acceptable semen quality in both breeding and non-breeding season. Furthermore, although seminal plasma composition was not a part of the current study, its composition during non-breeding may explain these differences (Domínguez et al., 2008) and has been reported to improve sperm frozen-thawed rate (Rickard et al., 2014;Kumar et al., 2014). Breeding season and non-breeding did not differ (p>0.05) for slow motility though breeding season had high VCL and VAP than non-breeding season. Temperature-humidity index has significant effect on physiological, biochemical, hormonal and enzymatic indices of indigenous sheep (Rathwa et al., 2017). Wobble spermatozoa was higher during non-breeding season than breeding season. Wobble is a comparison between average path and curvilinear (Mortimer, 1997). Therefore, spermatozoa collected during non-breeding season was moving unsteadily side to side.
Spermatozoa morphology and viability evaluation are ordered as normal, abnormal and live spermatozoa based on the set standards for characteristics evaluated on the constant scales: Loose head, loose tail, mid-piece, acrosome damage, distal droplets, bent tail and reacted acrosome (Björndahl, 2010). Live spermatozoa are vital to bypass cervix folds through crypt folds and to survive the selection and bio-fluid from the ewe reproductive tract (Fair et al., 2019;Rickard and de Graaf, 2020). It was not surprising to find high live spermatozoa during breeding season than non-breeding season, with the dead spermatozoa in the opposite direction in the current study. However, (Kadili et al., 2019) found high spermatozoa viability during non-breeding season than breeding season. Normal spermatozoa have a significant role in different ways. For example, sperm head is covered by the membranous sac contain nucleus and involved in transporting the DNA to the egg (Barbas and Mascarenhas, 2009). The membranous sac delivers the hydrolytic enzymes such as acrosin and hyaluronidase are released by exocytosis during fertilization to aid the spermatozoon to bind with zona pellucida during the socalled process of acrosome reaction. Conversely, loose head and coiled tails in this study were higher during breeding season than non-breeding season. These results were controversial to (Karagiannidis et al., 2000b;Barkawi et al., 2006;Francis et al., 2019).
No significant different (p>0.05) observed for midpiece abnormality, distal droplet, bent tail and reacted acrosome in this study. This implies that, irrespective of season, spermatozoa produced by Zulu rams are normal and the rams producing superior semen quality throughout the season. Ram that produce good quality semen throughout the year can be utilized for AI throughout the year (Al-Anazi et al., 2017) without hormone supplementation, to reduce costs of breeding. The sperm mid-piece is an energy power station containing mitochondria which is responsible for energy generation through a highly specialized Adenosine Triphosphate (ATP) (Barbas and Mascarenhas, 2009). As a result, it was remarkable to find non-significant different (p>0.05) for distal droplets in both seasons because retaining residual cytoplasm droplet correlate with Reactive Oxygen Species (ROS) that are normally produced by the glucose-6-phosphate dehydrogenase enzyme (Agarwal et al., 2003). There is an association between the membrane fatty acid profile and the acrosome reaction as well spermoocyte fusion (De Vriese and Christophe, 2003). Reacted acrosome could not differ among seasons. Therefore, non-significant different observed in the current study could meant that the sperm-oocyte fusion may not be affected by the season in Zulu sheep.
On the other hand, there was a noticeable gradual decrease of spermatozoa quality when the storage h' increases. For instance, after 24 h progressive motility numerically decreased by 16.09, 29.35% after 48 h and 33.26% at 72 h. These results were comparable to (Mata-Campuzano et al., 2015) and implies that, after 24 h spermatozoa are barely retaining motility hence, it is not recommended to inseminate with semen kept for more than 24 h (O'Hara et al., 2010) especial using cervical AI (Anel et al., 2005). D 'alessandro and Martemuci (2003), evaluated the seasonal effects on semen freezability and found that the highest postthawed survival was during summer and autumn (breeding season) due to seminal plasma protective effect (Domínguez et al., 2008). However, liquid stored semen represents an inexpensive alternative way to preserve endangered breeds through semen transfer from research stations to different farms for artificial insemination (Benmoula et al., 2017) than frozen state (Maxwell et al., 2007). In this regard, spermatozoa motility is a vital parameter extensively used to predict fertility (Hirano et al., 2001;Broekhuijse et al., 2012) after freezing and thawing (D'Alessandro and Martemuci, 2003). However, it is highly susceptible to the environmental changing condition such as excessive heat and or cold shock (Hafez and Hafez, 2000). Furthermore, have high metabolic activities after semen collection at the body temperature (37°C) hence, have less lifespan when not diluted (Salamon and Maxwell, 2000) and samples analysis should take place immediately after collection (Amann and Weberski, 2014). Therefore, in the current study it was expected that the progressive motility would be higher at 0 h than 24, 48 and 72 h. However, following semen-chilling, spermatozoa metabolic activities are suspended to prolong lifespan and activated after thawing (Salamon and Maxwell, 2000).
Non-progressive motility is inversely proportional to progressive motility (Holt et al., 2007). Therefore, when progressive motility decrease gradually as reported in the current study, non-progressive motility increase automatically at a similar pace. Hence, the increase of non-progressive motility as the storage period increases in the current study was determined by the increase of progressive motility and was expected.
The sum of progressive motility and non-progressive contributes to the total motility (Holt et al., 2007). In this study, total motility was greater at 0 h but steadily decreased as the storage period increases. These results were comparable to (Yániz et al., 2005;Sinha et al., 2019) where sperm motility and live sperm declined as preservation period increases. Nonetheless, contradicted to (Purdy, 2006;Purdy et al., 2010) on ram semen held at 5°C for 24 and 48 h prior cryopreservation, respectively. Numerically, Zulu ram semen in this study loosed 5% of total motility after 24 h of storage which is acceptable for cervical AI. Nevertheless, the ability of the spermatozoa to move through the cervical passage and mucus after natural mating or AI depends on rapid motility (Björndahl, 2010). Spermatozoa at 0 h was moving at relatively rapid motility (56.17±1.48) but prominently diminished as storage period increases to 24, 48 and 72 h though did not differ (p>0.05) between 48 and 72 h. These results were comparable to those reported by (Wang et al., 2015). Menchaca et al. (2005) obtained 43 and 35% motility when semen was stored on a tris based extender for 12 and 24 h respectively, at 5°C. In ram semen preserved for 48 h in MOPS titrated buffer, total motility did not differ from 0 h until 48 h. However, in tris based extender, drastically decline was observed (Yániz et al., 2011). Therefore, it might happen that, the decrease of motility (rapid and total motility) observed in the current study after 24 h was due to the less protecting effect from tris based extender (Albiaty et al., 2016). However, the effect extender might have in semen stored at 10°C still need more attention.
Curvilinear velocity in the current study was always higher than VSL and VAP though did not differ (p>0.05) between 0 h and 24 h as well between 48 h and 72 h. However, VCL at 24 h was higher than 48 h. These results were in line to (Yániz et al., 2011), where VCL in semen extended with tris based extender did not differ from 0 h until 24 h. The distance spermatozoa transit along its curvilinear path is called curvilinear velocity (Mortimer, 1997). Curvilinear velocity is a product from the sum of the distances along trajectory and corrected time. When male fertility is not compromised, curvilinear should be high followed by the average path velocity and the lowest being a straight-line velocity (Verstegen et al., 2002). Conversely, when male fertility is compromised, curvilinear tend to be a lowest and straight-line velocity value becomes the highest (Mortimer, 1997). The development of three velocity ratios allows advance explanations of the sperm trajectory (Mortimer, 1997;Holt et al., 2007). Spermatozoa velocity motions determines the velocity ratios and always expressed in the percentage form.
There is a close relationship between the Computer-Aided Sperm Analysis (CASA) motility (total and rapid motility) and in vitro fertilization rate in human (Hirano et al., 2001;Verstegen et al., 2002) and pigs (Hirai et al., 2001). Moreover, spermatozoa motility in sheep is associated with in vivo fertility and influence Artificial Insemination (AI) results (David et al., 2015). In the current study, total and rapid motility were higher during breeding season than non-breeding season. These results contradicted to (Benia et al., 2018) nonetheless, the method use might cause the differences since they used subjective method but were in line to (Kadili et al., 2019) who observed high total motility during autumn than winter in Beni Arous bucks. Other authors reported a seasonal effect on sheep reproduction efficiency where semen quality decreased (Zaragaza et al., 2009), depending on the latitude of origin (Carvajal-Serna et al., 2019). However, humidity and ambient temperature might cause negative effects (Ntemka et al., 2019).
Progressive motility determines the fertilizing ability of the sperm cell even after cryopreservation (Del Olmo et al., 2013). Progressive motility during non-breeding season differed (p<0.05) from the one during breeding season and so does with static and medium motility. These results were comparable to (Benmoula et al., 2017) in INRA180 rams, (Nitharwal et al., 2017), in Murrah buffalo, (Kadili et al., 2019) in Beni Arous bucks. Although fertility evaluation was out of the scope, these results might imply that, high spermatozoa population from non-breeding season will be able to pass cervix better than those from breeding season, due to the selection pressure cervix apply to the spermatozoa (Rickard et al., 2019;Rickard and de Graaf, 2020), selecting only viable spermatozoa with high progressive motility (Sakkas et al., 2015).
Only CASA parameters determines the velocity spermatozoa (VSL, VCL and VAP) (Hirai et al., 2001) and provide valuable physiological status of the spermatozoa (Broekhuijse et al., 2011). Spermatozoa velocities (VCL and VAP) are higher during breeding season (Kadili et al., 2019;Wang et al., 2015). Moreover, Sperm Velocities (VCL and VSL) can be used to determine fertile and infertile males (Hirai et al., 2001). In the current study, VCL and VAP was higher during breeding season than non-breeding season. Therefore, semen collected from breeding season can be regarded as more superior to the one during nonbreeding season. Although STR does indicator fertilization outcome in human being (Hirano et al., 2001), it did not differ (p>0.05) in the current study. The effect of season on semen parameters from several ram breeds have been previous reported (Chella et al., 2017;Benmoula et al., 2017;Badi et al., 2018). However, the present study is the first study reporting the seasonal variation in semen parameters from Zulu rams, assessed objectively and preserved at 10°C for up to 72 h.

Conclusion
These findings showed that semen parameters from Zulu rams does vary with season. Breeding season have better semen quality than non-breeding season. However, the differences observed cannot prevent Zulu rams from breeding during non-breeding season. Therefore, based on these results Zulu rams' can be classified as all year around breeders but studies involving fertility are still needed to verify these findings. On other hand, these results disclosed that preserving semen at 10°C result in a measly reduction of quality in both breeding and non-breeding seasons especially, when preservation period exceed 24 h until 48 h after that remain stable until 72 h. Therefore, Zulu ram semen stored at 10°C can retain quality until 72 h however, based on the spermatozoa quality obtained it is advisable to use the semen before storage period exceed 24 h in both seasons. Nevertheless, more studies are needed to test fertility and to look at the effect female may have when bred during non-breeding season and consider the feed effect when the lambing season take place during dry periods.

Acknowledgement
Special acknowledgement goes to Dr. FV Ramukhithi for technical assistance. Mr. TJ Mpofu is thanked for assisting in statistical analysis. Germplasm, Conservation and Reproductive Biotechnologies (GCRB) staff (Mr. M.L Mphaphathi and Ms. N Bovula) are acknowledged for their laboratory assistances.

Funding Information
National Research Foundation through Tshwane University of Technology. Agricultural Research Council, Irene through Germplasm, Conservation and Reproductive Biotechnologies (GCRB) unit for equipment, laboratories and consumables.

Authors' Contribution
Jabulani Nkululeko Ngcobo: Designed the study, collected and analyzed the data, as well wrote the manuscript.
Tshimangadzo Lucky Nedambale: Designed the study and revised the manuscript.
Khathutshelo Agree Nephawe: Designed the study and revised the manuscript.
Ayanda Maqhashu: Designed the study and revised the manuscript.

Ethics
This article is the original work containing unpublished material. The corresponding would like to confirm that all authors read and approved the final manuscript.