Post Thawing Sperm Quality and Ca
+2Intensity Characters of Local Goat Sperm
After Freezing by Simple Method Using Deep Freezing
Gatot Ciptadi1, Muhammad Nur Ihsan1, Sri Rahayu2, Sri Wahjuningsih1, Anis Mei Munazaroh1, Choirunil
Chotimah3, Irsanti Putri Ardyah 2 and R. P. Putra1 1Faculty of Animal Husbandry, University of Brawijaya 2Faculty of Mathematics and Natural Sciences, University of Brawijaya
3Central Laboratory of Biological Sciences, University of Brawijaya
Email: ciptadi@ub.ac.id
ABSTRACT
The objective of this research was to determine the effect of the simple modified freezing method, 1°C/minute freezing rate with different diluter ration on a post-thawing quality of local goat sperm namely Peranakan Etawah (PE). This work is aimed to study the quality of post-thawing sperm and to characterize the calcium intensity profile of both fresh and post thawing goat sperm. The method used is the experimental design of a laboratory. Freezing semen was performed in 2 main temperatures of -45°C then -196°C respectively using Mr. Frosty (®) System. Early Sperm characters of Ca+2 intensity was performed by Confocal Laser Scanning Microscope (CLSM) through Fluo-3 staining and Ca++ intensity was analysis descriptively. The result showed that post-thawing qualities are considered as good as standard qualities, at least, more than 40% based on Indonesian National Standard (SNI, 2014). The different level diluents commercial of Andromeda used were influenced highly significant (P<0.01). The best diluents ration is 1:4 (v/v) for final sperms stocked at -196°C. However freezing sperm conserved in -196°C is better than in -45°C. Meanwhile, the sperm characters of two condition showed the important variation of Ca+2 intensity, with the length of region measurement of 39.06±4.595 and 32.696±9.011 µm each. It was concluded that the calcium intensity pattern was varied more and higher in fresh
sperm than in freezing sperms. This simple modified method of a freezing system was considered as a feasible alternative method for goat semen in a reason for both for sperm post-thawing quality and practical purposes.
Keywords: Sperm; Local goat; Viability; Calcium Intensity; Artificial Insemination (AI).
INTRODUCTION
Artificial insemination (AI) is one way that is being implemented for local goats in Indonesia for the purpose of accelerating the improvement of the genetic quality and number of goat population. AI is done usually using frozen sperms (-196°C) of local goat superior sires selected, that are generally conducted with slow freezing (conventional).
The cryopreservation is known to disrupt the sperm plasma membrane and it induces premature capacitation of a sperm subpopulation, which may be a result of the increased internal calcium levels after thawing (Collin et al., 2000). It is well established that calcium plays an important role in the capacitation and then fertilization. In this study, we would like to test whether goat fresh sperm have different calcium levels intensity than sperm from freezing (post-thawing) sperm. Therefore, the aims of this research were to determine the effect of this modified conventional freezing method (-1°C/min) both for fresh and freezing sperms with different commercial diluter on post-thawing sperm quality and also to study their pattern of Ca+2 intensity.
The purpose of this study was to develop a modified simple method of freezing cells spermatozoa of Indonesia local goat known as PE in modified conventional system. This research was attempted to study the characters of the Ca+2 intensity of goat sperm after freezing by modified conventional method. Cormier et al. (1997) reported that calcium regulation by sperm from sires of low fertility appears to be deficient because their post-thawing sperms relative intracellular calcium level is higher than it is in bulls of good fertility. Ciptadi et al (2013) mentioned in oocyte fertilization by spermatozoa occurs an increasing in intracellular calcium concentration Over and over (Ca2+ Oscillations) as an early indicator the activation of oocyte.
MATERIAL AND METHODS
Animal used in this study was male goats aged 4 years and has been trained for the recovering and storage of semen by artificial vagina with a standard method. Semen prior to freezing for research, has previously been
quality tested by SNI with a minimum of 70% of individual motility. Each ejaculate was cryopreserved in commercial diluter according to standard procedures. For each experiment, 0.25 mL straws (40x106 sperm/straw) were thawed in a 37°C water bath for 60 seconds. Freezing was conducted with conventional modified methods with the speed (-1°C/min) with the help of Mr. Frosty system® and cryopreserved in -45°C using deep freezer then immersed in liquid nitrogen (LN2) at a temperature of -196°C, to do quality testing to post-thawing.
Method used was experimental design of Completely Research Designs one-way analysis, with main treatment of different level used commercial diluter (v/v). The treatments were dilution of commercial diluter: P1 (1:4), P2 (1:8) and P2 (1:3). Freezing semen was cryopreserved in 2 main final temperatures of -45°C then immersed in -196°C of liquid nitrogen, using Mr. Frosty (®) System. Early Sperm characters of Ca+2 intensity was performed by Confocal Laser Scanning Microscope (CLSM) through Fluo-3 staining and analysis descriptively. At least 5 sperms were selected to analyzed for each calcium intensity measure both for fresh and freezing sperms. The relative intracellular calcium intensity of each sperm was expressed as the line series of intensity pattern with help of CLSM images.
RESULTS AND DISCUSSION Post Thawing Sperm Quality.
Table 1. Post Thawing Sperm Quality of Local Goat Cryopreserved by Simple Method with Mr. Frosty, Deep Freezer and Stocked at -196°C.
Treatment of Dilution Level
Post Thawing Sperm Quality in Different Level of Dilution of Commercial Andromeda.
Motility (%) Viability of Live-dead cell (%) Abnormality cell (%)
P1 39.0+3.94 b 62.5+6.77c 5.6+1.07a
P2 21.0+3.16 b 33.5+4.12b 8.8+1.62a
P3 4.5+1.52 a 18.5+3.37a 18.5+3.24b
Note: different subscript in the same column mean highly significant (P<0.01).
The data on Table 1 showed that post-thawing qualities are considered as good as standard qualities, at least for P1 more than 40% (SNI, 2014). In this research with Mr. Frosty is considered as an equipment like a programmable freezing method, so it possible to produce the higher percentages of sperm motility, integrities of membrane and acrosome and viability when compared to the freezing method of manual during goat sperm freezing (Memon et al., 2013; Ciptadi et.al., 2016). The different diluents used were influenced highly significant (P<0.01) on all character of P2 and P3. The best diluents ration is P1, the 1:4 (v/v) for both final sperms stocked. However freezing sperm conserved in -196°C is better than -45°C (i.e. motility 39.3+9.4% vs. 56.0+5.6%). Deep freezer equipment for next future could be used as an alternative research development of freezing cell method. Purdy (2006), a cryopreservation protocol has been developed or optimized for sperm of one species, it may not be ideal for sperm of other species. Bovine and caprine sperm-freezing diluents, for example, contain similar ingredients, but interactions between goat seminal plasma and egg yolk are deleterious to the sperm, a situation not observed with bovine seminal plasma and egg yolk.
In this research, different ration of diluents is followed by lower cryoprotectant concentration. Kundu et al. (2000) reported that increase of the cryoprotectant of glycerol concentration caused a marked
decrease in motility. Changes in the cooling rate particularly before and during freezing had a notable effect on the sperm motility recovery. There was no or low recovery of sperm motility when the cells were transferred directly to liquid nitrogen from the initial two cooling stages. The data demonstrate the importance of all of the cooling stages in the cryopreservation of the cells
The Intensity of Calcium Intra Cellular. Confirmation of the intensity of intra-cellular calcium spermatozoa with CLSM showed that there was no change in the pattern of fresh semen and results of modified conventional freezing, but different in peal of Ca+2 intensity. The sperm conditions showed the important variation of Ca+2 intensity, with the length of region measurement of 39.06+4.595 um and 32.696+9.011 um each (Figure 1).
Figure 1. Different profile of calcium intensity of local goat sperm: (a) Fresh sperm and (b) Freezing sperms (post thawing) of local Indonesian Goat. It was analyses by Confocal Laser Scanning Microscope CLSM: 400, Z.4, 3 D.
Based on this research the calcium intensity pattern is varied more and higher in fresh sperm than freezing sperm (Figure 1). This result is in contraire with Bailey and Buhr (1994) that demonstrated that calcium levels are higher in cryopreserved sperm than they are in fresh semen. An interesting report demonstrated that a sperm subpopulation is capacitated as a result of cryopreservation even prior to artificial insemination (Cormier et al., 1997). Clearly, Ciptadi et al. (2016) noted that regulation of calcium plays is an important during capacitation and the acrosome reaction. On the basis that calcium regulation plays an important role during capacitation and acrosome reaction, both of which are necessary for successful fertilization, it is reasonable to speculate that intracellular sperm calcium levels are associated with semen fertility. Dragileva et al. (1999) Intracellular Ca2+ has a regulatory role in the control of sperm motility, capacitation, and the acrosome reaction. The use of Ca2+ by the cell as an intracellular messenger requires precise regulation of its intracellular concentration
CONCLUSIONS
This simple modified method of a freezing system was considered as a feasible alternative method for goat semen in a reason for both for sperm post-thawing quality and practical purposes. It is important to understand the specifics of sperm freezing method from a particular species i.e. local goat that will improve the cryosurvival of cell sperm. The calcium intensity pattern is varied more and higher in fresh sperm than freezing sperm. The results of this study indicate that the local goat sperm is frozen non-conventional Indonesia could be used for the application of artificial insemination. However, this method needs to be enhanced by using diluents and cryoprotectants more appropriate. It was necessary to study the relationship between the relative intracellular calcium intensity both fresh and freezing semen with their fertility in vitro.
ACKNOWLEDGEMENT
Ministry of Education Republic of Indonesia. Contract No. 253.102/UN10.21/PG/2016, 18 February 2016.
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