The present study investigated the effect of varying osmolarity of tris egg yolk citrate glycerol extender on seminal attributes of buffalo semen at various hours of refrigeration at 5 ºC. Twenty four ejaculates having mass motility ≥ 3+ from 4 bulls (6 ejaculate from each bull) were collected. Each ejaculate was divided into four group’s viz., Groups I, II, III and IV, diluted with extenders having osmolarities of 240, 260, 280 and 300 milliosmol/kg (mOs/kg), respectively, to 80×106 sperm/ml. After dilution, semen samples were filled in French mini straws and kept at 5°C and evaluated at 0, 24, 48 and 72 hours for various seminal attributes such as individual motility, viability, acrosomal integrity, and hypo-osmotic swelling test. No significant difference in individual motility at 0 hour was observed among all the groups. However at 24, 48 and 72 hours, the individual motility was significantly (P<0.05) higher in groups I and II as compared to groups III and IV. Other seminal attributes such as viability, acrosomal integrity and hypo-osmotic swelling test were significantly (P<0.05) higher in group II (260 mOsm/kg) as compared to other groups. It is concluded that seminal characteristics such as individual motility, viability, hypo-osmotic swelling (HOS) test and acrosomal integrity were significantly affected when the osmolarity of the extender was changed from 260 to 240, 280, and 300 mOsm/kg.
Artificial insemination (AI) and frozen semen technology played a vital role in improvement of milk production of the buffalo (Bubalis bubalis). From four to five decades, cryopreserved semen has been used for artificial insemination in the buffaloes, however, the conception rate is lower as compared to cattle (Barile, 2012; Anzar et al., 2003). It is presumed that sperm of buffalo are more prone to cryopreservation damages as compared to cattle and in general around 50% of buffalo sperm get damaged during freeze-thaw process leading to reduced motility, viability, acrosomal integrity, membrane integrity (Lone et al., 2016a), chromatin integrity (Khan and Ijaz, 2008; Lone et al., 2017), seminal antioxidants (Lone et al., 2016b) and increased concentration of sperm oxidants (Balamurugan et al., 2018; Lone et al., 2018) leading to reduced fertilizing potential of sperm. Dilution of semen with suitable buffer is of great importance for the maintenance of the fertilizing potential of the sperm (Rasul et al., 2000). Various extenders have been tried for semen preservation and tris (hydroxymethyl aminomethane) has successfully been used as an organic buffer for deep freezing of buffalo semen (Ijaz et al., 2009; Reddy et al., 2010; Lone et al., 2016c).
One of the crucial factors responsible for limited formation of ice crystals inside the cells is limited cell dehydration, and higher cell dehydration leads to increased sperm abnormalities (Agca et al., 2002). Thus the osmolarity modification of the extender may be a turning point as it determines the efflux of the water during freeze-thaw procedures (Iguer-Ouada and Verstegen, 2001; Aisen et al., 2002). Previously it was revealed that osmolarity of the semen may vary according to the season of the year and the variation in the osmolarity may affect semen quality in buffalo bulls during the various seasons of the year (Bhakat et al., 2015). Scanty information is available regarding the effect of osmolarity of extender on seminal characteristics of buffalo bulls during various hours of refrigeration. Hence the present experiment was aimed to study the effect of varying osmolarity of the tris extender on seminal attributes of buffalo bulls during refrigeration.
Materials and Methods
Semen was collected from four Murrah buffalo bulls, 4–6 year old, maintained at Artificial Breeding Research Centre (ABRC), ICAR-National Dairy Research Institute. The bulls were reared under the similar feeding and management conditions during the entire duration of the study.
Semen was collected during the morning hours using an artificial vagina as per the standard method. 24 ejaculates from 4 bulls (6 from each bull) were selected on the basis of mass activity (+3 and above) and individual progressive motility (70% and above). The mass activity of the semen sample was determined by assessing the motility of the spermatozoa following semen collection. Motility was graded on the scale of 0 to +5 (Tomar et al., 1966).
Semen Processing and Preservation
Immediately after ejaculate collection, it was divided into Group I, II, III and IV, diluted with Egg yolk-Tris-Glycerol (EYTG) extenders (Tris 24.22 g / L, citric acid monohydrate 13.6 g / L, fructose 10 g / L, benzyl penicillin 106 IU / L, streptomycin sulphate 1g / L, 20 % egg yolk, 6.4 % glycerol and millipore water added upto 1 L) to 80 million sperm / mL having osmolarities of 240, 260, 280 and 300, mOsm/kg, respectively. Osmolarity was determined by WECOR vapour pressure Osmometer (WESCOR model 5500, INC, USA), which was calibrated with 100-, 290- and 1000-mOsm standards and used to measure the osmolarity. After, dilution, semen samples were filled in French mini straws (0.25 mL) and kept at 5°C and evaluated at 0, 24, 48, and 72, hours for various seminal attributes such as individual motility, viability, acrosomal integrity, and hypo-osmotic swelling response. The details for the procedure of these parameters are given below-
Assessment of Sperm Motility and Live Sperm Count
For assessing sperm motility, a uniform drop of semen was placed on a clean grease free slide maintained at 37 °C. Then a clean cover slip was put on the semen drop and observation was done at 400× magnification of phase contrast microscope (Nikon Eclipse E600, Japan). The number of sperm moving in forward direction were estimated and expressed in percentage. In three different fields semen samples were analysed and a mean estimate of the three readings was taken as a final value of the sperm motility. Eosin-nigrosin stain was used to determine live sperm count as per the method described by Campbell et al. (1953). For preparation of stain 5 g of Eosin-Y (water soluble) and 10 g of Nigrosin were dissolved separately in 100 mL of 2.9% sodium citrate solution. The mixture was boiled for 15 min. The amount of volume which got evaporated during boiling was replaced with the buffer. Finally stain was prepared by mixing 10% Nigrosin solution and 5% Eosin-Y solution in the ratio of 4:1 in a container. The mixture was thoroughly shaken and filtered through Whatman filter paper. One drop of semen sample was mixed with three drops of stain and the semen-stain mixture was allowed to rest for about 1 min. After 1 min, a thin smear was prepared on a clean, grease free slide, air dried and then observed at 1000× magnification of phase contrast microscope. The sperm which appeared colourless or white were considered as live and those appeared partially or completely pink coloured were considered as dead. A total of 200 spermatozoa were counted in each slide and percentage of live sperm was determined.
Acrosomal Integrity and Hypo-Osmotic Swelling (HOS) Response
The acrosomal integrity was evaluated by Giemsa staining as per the method described by Watson (1975). After staining, to determine percent intact acrosome (PIA), a total of 200 sperm were counted at 1000× magnification of phase contrast. Functional integrity of sperm plasma membrane was assessed by the hypo-osmotic swelling test (HOST) as per the procedure described by Jeyendran et al. (1984) with some modifications. Briefly, to 100 µL of semen sample was added 1 mL of the HOST medium (4.9 g of sodium citrate and 9.9 g of fructose were dissolved in 1000 mL of distilled water) in an effendorf tube, followed by its incubation at 37 °C for 1 h. After incubation, a drop of semen sample was taken on a clean grease free slide and a cover slip was put and examined at 400× magnification. Sperm showing visible coiling of tail were considered as HOS responsive spermatozoa. To determine percent HOS responsive spermatozoa, a total of 200 spermatozoa were counted.
Individual motility was assessed subjectively under phase contrast microscope equipped with a warm stage (37 ºC) at 400× magnification. The live sperm percentage and abnormality was estimated by differential staining technique using Eosin-Nigrosin stain (Campbell et al., 1953). Acrosomal intactness was determined by Giemsa stain (Watson, 1975). Hypo-osmotic swelling test (HOST) was carried out according to the method described by Jeyendran et al. (1984).
Data were analyzed using one way ANOVA by Statistical Analysis System (SAS, 2011) Software Programme, version 9.3 and results were expressed as Mean ± SE.
Results and Discussion
The overall mean ±SE of various seminal attributes at 0, 24, 48 and 72 hours of refrigeration has been depicted in Table 1. No significant difference in individual motility at 0 hour was observed among all groups. However at 24, 48 and 72 hours, the individual motility was significantly (P<0.05) higher in groups I & II as compared to groups III & IV. Other seminal attributes such as viability, acrosomal integrity, and hypo-osmotic swelling response were significantly (P<0.05) higher in group II (260 mOsm/kg) as compared to other groups.
It has been seen that osmolarity of the extender influences the seminal characteristics; our study revealed that change in osmolarity of the dilutor affects the sperm characteristics of the buffalo semen during refrigeration which corroborates the findings of Mughal et al. (2017). Our study demonstrated that reduction in osmolarity from 300 to 260 mOsm/kg resulted in improvement in various seminal characteristics such as motility, viability, HOS response and acrosomal integrity, however when the osmolarity was reduced to 240 mOsm/kg, there was a significant reduction in motility, viability, HOS response and acrosomal integrity. Previous studies have revealed that there was significant reduction in motility, viability, HOS response, acrosomal integrity and increased DNA damage, when the osmolarity of the extender was reduced from 275 to 255 mOsm/kg (Mughal et al., 2017).
Table 1: Seminal attributes of buffalo semen diluted with extenders of various osmolarities at various hours of refrigeration (Mean ± SE)
|Seminal Attributes (%)||Period||Group I||Group II||Group III||Group IV|
|(240 mOsm/kg)||(260 mOsm/kg)||(280 mOsm/kg)||(300 mOsm/kg)|
|Individual motility||0 h||69.56a±0.26||69.70a±0.26||68.43a±0.26||69.48a±0.26|
|HOS Response||0 h||63.29ac±0.24||63.70b±0.24||63.06c±0.24||62.99c±0.24|
Means bearing different superscripts within the same row differed significantly (P<0.05).
The plasma membrane surrounding the sperm holds various organelles and intracellular components and maintains chemical gradient of ions and soluble components. Plasma membrane also has got some specific proteins that are involved in the transport of ions or components (Schurmann et al., 2002). So any change in the integrity of the plasma membrane may lead to sperm death or reducing fertility level of the sperm. Buffalo spermatozoa revealed better semen quality after dilution and cryopreservation, when the semen was diuted with the extender of osmolarity of 260 mOsm/kg. In frozen-thawed bull semen, highest percentage of motile spermatozoa was found when semen was diluted with extenders having osmolarities ranging from 270 and 340 mOsm/kg (Liu et al., 1998b). Due to solute loading and outpouring of water phenomena, it is better that extenders should be towards hypotonic side in terms of osmolarity as compared to hypertonic side. Maintaining osmolarity of medium as 300 mOsm/kg in bovine semen yielded better results in terms of membrane integrity and sperm motility (De Pauw et al., 2003). Percentage of motile spermatozoa declined sharply, when the osmolarity of the modified Tyrode’s solution was increased to >500 mOsm (Liu and Foote, 1998). Dilutors needed for semen preservation are designed in such a way that they should be nearly isotonic to semen, in order to reduce the effects on metabolism and survival of spermatozoa (Bredderman and Foote, 1969). Osmolarity environment is a vital factor that needs to be considered during cryopreservation of semen. Anisoosmotic solutions not only lead to induction of stress in cells, but also determine outcome of process of cryopreservation along with the freezing/thawing processes, there are important osmotic changes due to differential freezing medium, leading to flux of water through the cell membrane (Curry and Watson, 1994).
It is concluded that buffalo sperm are sensitive to the osmotic pressure of the extender used for dilution of semen. Seminal characteristics such as individual motility, viability, hypo-osmotic swelling (HOS) response and acrosomal integrity were significantly affected when the osmolarity of the extender was changed from 260 to 240, 280, and 300, mOsm/kg. A marked change in the osmotic pressure of the semen dilutor may alter freezability and fertility of buffalo sperm. It is recommended that osmolarity of the dilutor used for preservation of buffalo semen at refrigeration temperature should be closer to the osmolarity of the seminal plasma.
The authors are thankful to the Director and Vice-Chancellor of ICAR-National Dairy Research Institute, Karnal for providing the facilities and Indian Council of Agricultural Research, New Delhi, for an award of a senior research fellowship for Ph.D. program to the first author.
Conflict of Interest
The authors declare that they don’t have any conflict of interest.