Preservation of goat semen at 5°C with emphasis on its freezability and the impact of melatonin

Evaluation of goat semen, extended in Cornell University 16 extender, was used to study the effects of melatonin of various concentrations on sperm motility (SM) %, alive sperm (AS) % and sperm abnormalities (SA) % of extended goat semen stored at 5°C for seven days were studied. Melatonin at the doses of 0.0, 10.0, 15.0 and 20.0 μg/ 100 x 106 sperm was added to the extended semen and the samples were examined daily for the previous parameters. Additionally, the infl uence of melatonin on post-thawing motility was assessed. The results of the present investigation elaborated that melatonin particularly the high concentration (20.0 μg) signifi cantly improved SM%, reduced dead sperm % and improved post-thaw sperm motility. In conclusion, the addition of melatonin (particularly 20.0 μg) induced remarkable and profound physiological actions on goat semen quality during storage for long time at 5°C and improved its freezability during preservation. Research Article Preservation of goat semen at 5°C with emphasis on its freezability and the impact of melatonin El-Battawy KA* Department of Animal Reproduction and Artifi cial Insemination, National Research Center, Egypt Received: 11 June, 2019 Accepted: 09 July, 2019 Published: 10 July, 2019 *Corresponding author: El-Battawy KA, Department of Animal Reproduction and Artifi cial Insemination, National Research Center, Egypt, E-mail: https://www.peertechz.com


Introduction
The success of an artifi cial insemination (AI) program depends on the proper management of semen collection, storage and use. Goat semen has been stored at temperatures ranging from 2 to 15°C and mostly at 5°C using various diluents such as sodium citrate-yolk, sodium citrate-fructose-yolk, milk (whole, skimmed or reconstituted) with or without egg yolk, Spermasol, Neoseminan, saline [1]. It was found that trisbuffer extenders were effective for dilution and storage of goat, sheep and rabbit semen [2][3][4].
Nishikawa et al., [5], stored goat spermatozoa in Neoseminan diluent at 4°C for 8-15 days and maintained their fertilizing capacity for 6 days. Moreover, Eppleston et al., [6], demonstrated that goat spermatozoa have retained their fertilizing capacity for at least 8 days of storage at 5°C in Tris-Fructose-citric acid-yolk diluent. Unfortunately, there is no available literature regarding the use of glycine containing extenders to keep the viability of goat semen. On the other hand, the replacement of citrate buffer with glycine was reported to improve the survival of ram spermatozoa. Also, El-Chahidi [7][8][9], elaborated that the use of different combinations of electrolytes mixture and glycine improved the motility and viability of ram spermatozoa for long time.
Moreover, the use of glycine and glucose containing extenders maintain high fertility over a period of several days in bull [10].
Melatonin (N-acetyle-5-Methoxytryptamine) is a hormone produced mainly by the pineal gland besides other tissues like retina [11]. It neutralizes free radicals thus it prevents cell damage. Mammdouh et al., [12,13], recorded that the addition of melatonin to liquid bull semen improved its storage and preservation for 6 days. Unfortunately, no available literature was found concerning the addition of melatonin to goat semen.
Thus the objective of the present study is to: 1. Study the effect of some extenders on viability of goat semen during preservation at 5°C.
2. Study the physiological infl uence of melatonin on the quality of goat semen preserved in liquid or frozen condition.

Materials and Methods
This investigation was carried out at private goat farm at Alexandria desert road.

B-experimental materials
Melatonin was imported from Twinlab Specialty Corporation, Ronkon Koma, New York, USA. While, the other chemical reagents used for the preparation of extenders were purchased from Sigma-Aldrich Co., Deisenhofen, Germany.

C-semen collection and evaluation. At 1000 rpm for ten minutes
Semen was collected twice weekly by means of an artifi cial vagina using an anestrous doe as a teaser for a period of two months. After collections, the ejaculates were transferred to the laboratory of the farm within 2-3 minutes where they were centrifuged to remove the seminal plasma and consequently to avoid the harmful effect of seminal goat enzyme. Semen was kept in water bath at 30°C for evaluation by means of conventional methods.

E-semen processing and experimental design
Only ejaculates of >70% initial motility and 2000 x 106 sperm cells/ ml were used in the following experiments: Experiment I: This experiment was designed to fi nd out the impact of melatonin on the viability of chilled goat semen in CU-16 extender. Semen samples were divided and diluted (1: 4 according to Evans and Maxwell, [3], at 30°C with semen extenders. After dilution, the extended semen was incubated at 5°C to be examined daily for seven days for SM %, AS % and SA % using eosin-aniline stain according to Shaffer and Almquist [15]. Melatonin was added to the extended semen at the rate of 0.0 μg (control sample), 10, 15 and 20 μg/100x106 sperm [13].

Experiment 2:
This experiment was designed to fi nd out the impact of melatonin on the viability of chilled goat semen in CU-16 extender as it achieved the best results in comparison with the rest of used extenders. Semen samples were processed as previously mentioned in experiment I, then melatonin was added to the extended semen at the rate of 0.0 μg (control sample), 10, 15 and 20 μg/100x10 6 sperm [13]. After daily examination, the samples were centrifuged at 3000 rpm for 20 minutes to get supernatant for immediate daily determination of acid phosphatase (AcP) according to Moss [16].

Experiment 3:
This experiment was designed to investigate infl uence of melatonin (0.0, 10, 15 and 20 μg) on freezability of goat semen exte d in TGGY, TFGY, CU-16 and 14 extenders. Semen samples were split and diluted 1 : 4 at 30 °C. The diluted semen was cooled and loaded into 0.25 ml straws at 5°C. The straws were placed horizontally on freezing racks and lowered into liquid nitrogen vapour inside small tank containing 10 liters of liquid nitrogen at a height of 2.0 cm above the level of liquid nitrogen, for 15 minutes. The straws were then immersed gradually (within 2-3 minutes) in liquid nitrogen and transferred into liquid nitrogen storage container. After few weeks, frozen goat semen was thawed in a water bath at 40°C for 30 seconds. The thawed semen was emptied in pre-warmed tubes and incubated in water bath at 30°C for assessment of sperm motility [17].

F-statistical analysis
Data were transformed from percentage to absolute fi gures using arcsin tables. The ANOVA test was used at a confi dence not less than limit 95% using SAS program (1988). LSD test was used to evaluate the signifi cant difference between means at P<0.05. Table 1 declared the signifi cant (P<0.0001) effect of melatonin on sperm motility %. Furthermore, both Table 2 and  Table 3 showed the infl uence of melatonin on both AS % and SA %.

Results
The high concentration of melatonin (20 μg) had signifi cantly (P<0.0001) increased the motility and the live sperm in comparison with the other concentrations, during  (Table 4).

Discussion
This investigation elaborates to effect of various extenders on preservation of extended goat semen, impact of melatonin on sperm motility %, alive sperm %, sperm abnormalities % as well as freezability of that semen. It was noticed that the use of TGGY, TFGY, CU-16 and 14 extenders improved signifi cantly the storage of extended semen. These results are compatible with Evans and Maxwell [3], Roca et al., [4] and Chehadeh et al., [2], who reported that Tris-buffer extenders were the best diluents for sheep, rabbit and goat respectively. Regarding the storage of goat semen in glycine containing extenders (CU-16 and 14), our results are in agreement with Shannon [10] and El-Chahidi [9], who reported that the inclusion of glycine in extender improved and allowed long time storage of bull and sheep semen respectively. On the contrary Dessouky et al., [18], found that the glycine containing extenders were less effi cient in storage of rams semen Paleg et al., [19], attributed the benefi cial effect of glycine to its ability to retard thermal denaturation of enzymes thus it maintains the enzyme structure and function via its protective action.
It was observed that addition of melatonin resulted in a signifi cant increase in sperm motility and alive sperm percentages in all types of extenders. The effects of melatonin as an additive were profound and clear in high concentrations and at the last four days of incubation. These results are in accord with Mamdouh et al., [13], who reported the same results on addition of melatonin to liquid bull semen. On the other hand, our results were in disagreement with Bornmann et al., [20], who concluded that seminal plasma melatonin play no important role in sperm motility. The infl uence of melatonin on sperm motility and alive sperm percentages may be ascribed to one and/or all of the following physiological mechanisms: I-Melatonin increases ATP ase levels [21]. The increase of ATPase is correlated with an increase in ATP which is the main energy source used by the sperm fl agellum to initiate and activate forward motility [22].
III-Melatoni could be a potent cyclic AMP (cAMP) stimulator [24]. cAMP stimulates sperm motility via its direct action on the axoneme of the tail [25] or indirectly through acting on the cell membrane as secondary messenger [26].
Also, it was obvious that melatonin induced a signifi cant decrease in the sperm abnormalities percentages and a signifi cant reduction in seminal acid phoshatase. These results were in accord with Abdine [27], who recorded that melatonin treatment of Cambridge rams, in vivo, lowered the sperm dead percentage and decreased the abnormal sperms. Additionally, our results were coincided with Poeggeler et al., [12], who reported that the number of abnormal and dead sperms was reduced after addition of melatonin. Moreover, the current results were in a harmony with those results obtained by Mamdouh et al., [13], who concluded that melatonin decreased signifi cantly sperm abnormalities and acid phosphatase level. The impact of melatonin on sperm abnormalities and acid phosphatase could be accredited to one and/or the following physiological actions: 1-Melatonin can pass the cell membrane and protects DNA from free radical damage effect through its potent antioxidant and anti-aging effects on the cells [12].   At last, it was clear that melatonin produced signifi cant increase in post-thawing motility. These results were in agreement with Kaya et al., [28], who concluded that melatonin administration to rams, in vivo, improved post-thawed sperm viability as well as the intact acrosome rates. The benefi cial effect of melatonin on post-thawed motility could be attributed to its decreasing effect on the phosphatase enzyme release (leakage) from sperm cells during cryopreservation [29].
In conclusion, the addition of melatonin (particularly 20.0 μg/100 x 106 goat sperm) induced remarkable and profound physiological actions that improved the extended goat semen quality, its storage for long time at 5°C and improve its freezability.