Background: Sperm cells are really sensitive to environmental alterations, particularly pH, osmolarity and temperature. To protect the sperm during some long or short clip uses like cryopreservation or micromanipulations, it is indispensable to command the environmental conditions. Aim: The aims of the present survey were to happen the effects of different osmolarities of an ionic-based medium ( modified Tyrode ‘s solution ) and a non-ionic based medium ( milk ) on the bull sperm during incubation at above zero temperatures. Material and Methods: First experiment: Semens were collected ( from five Najdi bull ) , centrifuged and most parts of seminal plasma was removed. The staying concentrated seeds were splited into nine aliquots to incubate in three different osmolarities ( 200, 300 and 400 mOsm ) at three incubation temperatures ( 5, 25 and 39A°C ) . The aliquots were assorted with same volume of different tyrode ‘s osmolarities ( 100, 300 and 500 mOsm ) to hold a suspension with 200, 300 and 400 mOsm. The sperms of each aliquot was analyzed after 15 and 60 min of incubation. Sperm viability and progressive motility were analyzed and compared amongst different groups. Second experiment: Semen was collected from same males from the first experiment and assorted with the same volume of whole cow milk ( at 5 and 25A°C ) and milk with 7 % glycerin ( at 5A°C ) for 15 and 60 min. Consequences: Sperm motility badly affected ( P & lt ; 0.05 ) by incubation at low ionic tenseness ( 200 mOsm/l ) particularly at low temperature ( 5A°C ) . The impact of low osmolarity on sperm viability may be reduced by increasing the temperature of incubation to 39A°C. The reduced sperm motility, which was induced by take downing osmolarity, was non improved ( P & gt ; 0.05 ) by increasing temperature during 1 H of incubation. The 2nd experiment has shown that milk can protect the sperm viability and motility at cool conditions and there is no good consequence of glycerin in combination of milk on sperm viability and motility at above zero temperatures ( P & lt ; 0.05 ) . Decision: Iso- and hyper-osmotic solutions can protect the sperm motility and viability at 25 and 39A°C, while non-ionic solutions ( milk ) can utilize for protection of sperm at 5A°C incubation.
Cardinal words: Bull sperm ; Tyrode ‘s solution ; Whole milk ; Osmolarity ; Glycerol
Introduction
Sperm is exposed to different medium osmolarities and temperatures during freeze or use. Cryoprotectants are the high molecular weight substances like glycerin, ethylene ethanediol or di methyl sulfoxide etc. , and that are cytotoxic at high temperatures. After exposing to the cryoprotectants at low temperatures, the intracellular H2O is excluded from the sperm and the osmolarity of the intracellular environment increased, this consequence is besides really unsafe for the sperm, which is called “ solution effects ” ( 1 ) . Therefore, the cryopreservation protocol should be able to cut down these hazard factors: take downing the temperature ( from 38 to -196A°C ) and toxic effects of the cryoprotectants. Cooling rate, equilibration clip and temperature are three of import factors in sperm freeze plans. Impact of chilling rate on sperm motility may be mediated by alterations in osmolarity ( 2 ) .
On the other manus, incubating the sperm at above zero temperatures for short clip transit for unreal insemination in some domestic carnal species and it ‘s use during human sperm processing have been considered. Effectss of non-ionic and ionic dilutants on sperm incubation at above nothing temperatures have been investigated in different species ( 3,4,5 ) . Some surveies reported the structural abnormalcies of sperm after exposing in pervading ( 6 ) and ionic-based ( 4 ) solutions at above zero temperatures. Liu and Foote ( 1997 ) have shown that the best bull sperm reaction for above zero temperatures incubation is dependent on the medium osmolarity, temperature and clip of incubation ( 7 ) . Rigby et al. , ( 2001 ) reported the good effects of modified Tyrode ‘s solution for continuing the entire sperm merely when seminal plasma was be removed ( 5 ) . Increased Deoxyribonucleic acid harm due to chilling bull sperm in skim-milk egg yolk at above nothing temperature has been reported, excessively ( 8 ) . These impact of osmolarities on the sperm parametric quantities may be influenced by the composing and temperature of medium ( 7 ) , species ( 9 ) , persons ( 5,10 ) and the beginnings of gathered sperm ( 11 ) . For many old ages, research workers have reduced the amendss caused by stop deading by the add-on of natural protein and lipoprotein beginnings like egg yolk or milk in combination with the cryoprotectants ( 1 ) . The good consequence of egg yolk for the saving of cooled sperm has besides been documented ( 7 ) . There are studies of protecting effects of the milk based media on sperm motility during storage ( 8,12 ) . The purposes of the present survey are to measure the sperms reaction after exposing it to different osmolarities of Tyrode ‘s solution and milk at different temperatures during two different incubation times.
Material and methods
Bull Semen
Semen was provided from bulls ( n=5 ) at the research centre for Agriculture and Natural Resources of the Khuzestan state, Iran. Semen was collected utilizing an unreal vagina ( 39A°C ) and transported to the research lab in a visible radiation protected 35A C flask for 1 hr.
Media readying
Modified Tyrode ‘s solution ( in g/l 8.18 NaCl, 0.24 KCl, 0.04 NaH2PO4, 2.18 NaHCO3, 0.08 MgCl2 6H20, 0.31 CaCl2 2H2O, 0.94 glucose ( All from Merk, Germany ) , and 2.48 HEPES ( Sigma, USA ) : 300 mOsm/l ) was the basic medium in experiment 1. The HEPES buffer was used to forestall the rapid alterations in the medium pH. The modified Tyrode ‘s solution with 100 mOsm/l was prepared by thining 1 portion of Tyrode ‘s with 2 parts of deionized H2O. To supply 500 mOsm/l solution of Tyrode ‘s, all values pf chemical constituents ( without HEPES ) of Tyrode ‘s were multiplied by 1.66 and added to 1 litre of deionized H2O ( 7 ) . Osmolarities of all solutions were measured and confirmed by a graduated vapour force per unit area osmometer ( Model 5500, Wescor, Logan, UT, USA ) . Whole cow milk ( Choopan dairy industries, Iran ) has been heated ( to 95A°C in boiling H2O over 30 to 45 min. ) to demobilize lactenin in the protein fraction because of its toxicity ( 13 ) . The het whole milk used entirely or was supplemented with 7 % glycerin ( Merk, Germany ) for the 2nd experiment. All of the media in this survey were supplemented with 0.25 mg/ml of gentamycin ( Daru-pakhsh, Iran ) .
Extension of Semens
Fresh seeds samples were centrifuged at 500 g for 25 min to take most of the seminal plasma to supply the sperm concentration of about 6 A- 109/ml. The concentrated seed was assorted 1:1 ( v/v ) with assorted media by soft stirring, giving a concluding sperm concentration of about 3 A- 109/ml. As dilutants and seeds were combined in equal volumes, the concluding osmolarities of the suspensions of sperm were the agencies of the osmolarities of different media with that of seeds ( about 300 mOsm/l ) . All the processs of seeds extension were performed at the room temperature.
Sperm analysis
An adept individual in this field of survey undertook the sperm analysis processs. The per centum of motile and feasible sperm was estimated 15 to 60 min after initial exposure to the media. Before rating, samples were subjected to iso-osmotic modified Tyrode ‘s solution. A portion of sperm was assorted with a portion of Eosin B/Nigrosin discoloration ( Sigma, USA ) on a slide for 1 min and dried by puting it on a hot plate. A sum of 100 sperm per sample was counted with the light microscopy at a magnification of 100. Unstained sperms were considered to be feasible ( 14 ) . Sperm progressive motility was estimated by puting a little volume of sperm suspensions, covered by a coverslip on a heat phase under a light microscopy.
Experiment 1: Sperm in Tyrode ‘s solution varying in osmolarity
The volume of sperm ( n=5 ) in Tyrode ‘s solution was measured in a factorial experiment with three osmolarities of the Tyrode ‘s: seeds combination ( 200, 300, and 400 mOsm/l ) , and examined after two different incubation times ( 15 and 60 min ) at 5, 25 and 39A°C and replicated with five seeds samples from different bulls. The sperm suspension aliquots were placed in a water-jacketed microtube. Then the water-jacketed microtubes were placed in the icebox ( 5A°C ) , room temperature ( 25A°C ) or a warm H2O bath ( 39A°C ) .
Experiments 2: Sperm in milk with and without 7 % glycerin
Equal volumes of seeds samples ( n=5 ) were assorted with milk without glycerin at two temperatures ( 5 and 25A°C ) and milk with 7 % glycerin ( at 5A°C ) . Progressive motility and viability of sperms were evaluated after 15 and 60 min of incubation. The experiment was replicated with five seeds samples from different bulls.
Statistical analysis
In the first experiment, effects of osmolarity, temperature and continuance of incubation and their interaction on the per centums of feasible and progressive motile sperms, in a split secret plan design, were analyzed by ANOVA utilizing General Linear Model ( GLM ) process of SAS ( the chief secret plan was the medium osmolarities and two subplots were the temperatures and the continuances of incubation ) . In the 2nd split secret plan design, the effects of dilutants ( milk with and without glycerin ) through two times of incubation at different temperatures on the per centums of feasible and progressive motile sperms were analyzed utilizing GLM process of SAS. Least square agencies ( LSmeans ) were compared with pdiff trial in SAS ( 15 ) . Datas are presented as the LSmeans A±standard mistake of agencies ( SEM ) .
Consequences
Experiment 1:
The consequences are presented in figures 1 and 2. Entire sperm viability decreased after 60 min ( 51.4A±3.02 % ) comparison to 15 min of incubation ( 60.8A±3.01 ; P & lt ; 0.05 ) . The per centums of feasible sperm were 43.4A±3.6, 56.3A±3.9 and 68.6A±3.6 at 5, 25 and 39A°C incubation, severally ( P & lt ; 0.05 ) . Sperm viability significantly decreased ( P & lt ; 0.05 ) by incubation in 200 mOsm ( 30.2A±3.7 medium ) comparison to the 300 ( 72.2A±3.7 ) and 400 ( 65.8A±3.7 ) mOsm media. However, the per centum of feasible sperms was non different between 300 and 400 mOsm ( P & gt ; 0.05 ) media. While there was non ( P & gt ; 0.05 ) any interaction between continuance of incubation, temperature and medium osmolarity ( Fig. 1 ) , a important ( P & lt ; 0.05 ) interaction was observed between medium osmolarity and incubation temperature on sperm viability. Medium osmolarities of 300 and 400 mOsm/l, at 5A°C, ( 53.8A±6.5 vs. 48.1A±6.05 ; P & gt ; 0.05, severally ) significantly ( P & lt ; 0.05 ) reduced the impact low osmolarity ( 200 mOsm/l ) on the per centum of feasible sperms ( 23.7A±6.05 ) . The highest per centum of feasible sperms, at 25A°C, was observed ( P & lt ; 0.05 ) in 300 mOsm/l medium ( 83.8A±6.5 ) comparison to 200 and 400 mOsm/l media ( 22.5A±6.5 vs. 62.5A±6.9 ; P & lt ; 0.05, severally ) . Although, incubation at 39A°C ( 44.4A±6.5 ) reduced ( P & lt ; 0.05 ) the impact of 200 mOsm/l medium at 25 and 5A°C on sperm viability ( % ) , higher osmolarities ( 300 and 400 mOsm/l ) are required ( P & lt ; 0.05 ) to optimise the per centum of feasible sperm ( 74.5A±6.05 vs. 86.9A±6.05 ; P & gt ; 0.05 severally ) at 39A°C. The forms of alterations were similar at two different ( 15 and 30 min ) continuances of incubation ( Fig. 1 ) .
There was no difference between 15 ( 37.5A±3.6 % ) and 60 ( 37.8A±3.6 % ) min of incubation on sperm progressive motility ( P & gt ; 0.05 ) . Entire sperm progressive motility ( % ) decreased ( P & lt ; 0.05 ) at 5A°C ( 22.1A±4.3 ) comparison to 25 and 39A°C ( 41A±4.5 and 49.8A±4.4 ; P & gt ; 0.05, severally ) . Overall sperm imperfect motilities ( % ) were 12.9A±4.4, 53.01A±4.4 and 47.1A±4.4 at 200, 300 and 400 mOsm media, severally ( P & lt ; 0.05 ) . While there was non ( P & gt ; 0.05 ) any interaction between continuance of incubation, temperature and medium osmolarity ( Fig. 2 ) , a important ( P & lt ; 0.05 ) interaction was observed between medium osmolarity and incubation temperature on sperm motility ( % ) . The highest ( P & lt ; 0.05 ) sperm motility ( % ) at 5A°C was observed in 300 mOsm/l ( 31.3A±7.6 ) comparison to 200 and 400 mOsm/l solutions ( 12A±7.6 vs. 23.1A±7.4 ; P & lt ; 0.05, severally ) . The highest ( P & lt ; 0.05 ) sperm motility at 25A°C was observed in 300 mOsm/l solution ( 71.1A±7.6 ) comparison to 200 and 400 mOsm/l ( 5.3A±7.6 vs. 46.7A±8.1 ; P & lt ; 0.05, severally ) solutions. The highest sperm motility ( P & lt ; 0.05 ) at 39A°C was in 400 mOsm/l ( 71.5A±7.6 ) comparison to 300 ( 56.7A±7.6 ) and 200 ( 21.25A±7.6 ) mOsm/l solutions. The forms of alterations were similar at two different ( 15 and 30 min ) continuances of incubation ( Fig. 2 ) .
Experiment 2:
The consequences of this experiment are presented in the tabular arraies 1 and 2. There was no interaction between the continuance of incubation, temperature and the composing of dilutants on sperm viability and progressive motility ( P & gt ; 0.05 ) . Entire per centums of unrecorded sperms were 67.6A±6.3 and 52.2A±6.3 after 15 and 60 min of incubation ( P & gt ; 0.05 ) , severally. Entire sperm viability ( % ) was non different ( P & gt ; 0.05 ) in milk at 25A°C ( 70.5A±7.8 ) , and 5A°C ( 54.5A±7.8 ) and milk with glycerin at 5A°C ( 54.6A±7.8 ) . The forms of sperm viability alterations in different media were similar at two different incubation times ( Table 1 ; P & gt ; 0.05 ) .
Entire sperm progressive motilities ( % ) were 34.6A±7.4 and 18.8A±7.4 after 15 and 60 min of incubation, severally ( P & lt ; 0.05 ) . The per centum of progressive motile sperms was non significantly different ( P & gt ; 0.05 ) between milk at 25A°C ( 32A±9.1 ) , milk at 5A°C ( 24.2A±9.1 ) and milk with glycerin at 5A°C ( 23.7A±9.1 ) . The forms of sperm progressive motility alterations in different media were similar at two different incubation times ( Table 2 ; P & gt ; 0.05 ) .
Discussion
Incubation of sperm in an ionic base solution at low temperature decreased motility and viability of sperm particularly under status of lower ionic tenseness ( 200 mOsm/l ) . Higher osmolarities increased sperm viability and motility in additive manner thorough the temperature alterations at least up to 1 H of incubation. Datas have shown that sperm motility increased significantly at 39A°C, the conditions similar to the process for sperm capacitation during IVF ( 16 ) . Using the Tyrode ‘s solution ( 5 ) and supplementation of storage media with modified Tyrode ‘s solution ( 17 ) were effectual for sperm protection during liquid sperm storage. From a old survey, Liu and Foote ( 1997 ) have shown important alterations of sperm motility in the 200 and 400 mOsm/l Tyrode ‘s base solutions. When they adjusted the osmolarities by NaCl alterations, they found many nonmotile sperms with integral plasma membrane at higher osmolarities ( 7 ) .
The 2nd experiment has shown the good effects of whole milk base dilutant for saving of sperm at above zero temperatures particularly at cool storage for at least 1 hr. Egg yolk and milk improved the end product of sperm cryopreservation. Foote and Liu ( 1997 ) found that solution incorporating egg yolk ( 300 mOsm/l ) protected the sperm motility during 1 H of cool storage ( 7 ) . Data of the present survey besides confirms that utilizing whole milk can cut down the impact of take downing temperature on sperm viability and motility. The good consequence of milk is limited to the cool temperature and at the higher temperatures, it can non protect the sperm viability and motility. The mechanism by which milk protect the sperm surface is different from egg yolk, as protection appear to shack with the protein fraction instead than lipoprotein or phospholipids ( 13,18 ) . Batellier et Al. ( 2001 ) showed that while some milk fractions ( ultrafiltrate, microfiltrate, and a-lactalbumin fraction ) decreased spermatozoal endurance, others ( beta-lactoglobulin and native phosphocaseinate ) were protective ( 19 ) .
Comparing egg yolk and skim milk based extenders with the Ringer solution, Andrade et al. , ( 2008 ) showed that the sperm motility was better preserved during the incubation from 30 to120 proceedingss for the seeds diluted in yolk egg-citrate and skim milk-based extenders than from the Ringer extender ( 4 ) .
Sperm sensitiveness to the osmotic alterations was studied in many different species. Equine sperm is immune to osmotic daze ( 9 ) . There are single fluctuations in bull ( 10 ) and mouse ( 6 ) sperm opposition to osmotic daze. Beginning of the provided sperm may besides hold a important consequence on the osmotic behaviour of sperm ; epididymal sperm is more immune than ejaculated sperm against osmotic emphasis ( 11 ) . Impact of osmotic emphasis on sperm can be mediated thorough an oxidative emphasis ( 20 ) . Cooling can besides exercise its consequence on sperm thorough osmolarity alterations ( 2 ) . Zhang et Al. ( 2001 ) reported alterations in sperm motility in a temperature-time-medium dependent relation. They found that glycin betaein can better the sperm motility at lower temperatures, with no consequence at 37A°C ( 21 ) . Zhou et Al. ( 2004 ) concluded that Poly Vinyl Alcohol ( PVA ) can be used to replace for Bovine Serum Albumin ( BSA ) and 20A°C is more suited than 15A°C for Sus scrofa seeds storage, and in vitro fertilizing capacity of sperm cell is maintained for at least 8 yearss in Zorlesco+PVA at 20A°C ( 22 ) . Li et Al. ( 2006 ) show that incubating bovid sperm with cholesterol-loaded cyclodextrin ( CLC ) before cryopreservation increases the per centums of motile and feasible cells recovered after stop deading and dissolving ( 23 ) . The optimum conditions for saving of mouse sperm cell were 800 mOsmol KSOM incorporating 4 mg/ml BSA and a keeping temperature of 4A°C ( 24 ) .
In decision, Iso- and hyper-osmotic solutions can protect the sperm motility and viability at 25 and 39A°C, while non-ionic solutions ( milk ) can utilize for protection of sperm at 5A°C incubation.
Recognitions
This survey was funded by Shahid Chamran University of Ahvaz and Khuzestan Organization of Jihad-Agriculture, Ahvaz, Iran.
Table Legends
Table 1. Effectss of different media ( Milk at 5A°C and 25A°C, Milk with glycerin at 5A°C ) during two different incubation times ( 15 and 60 min ) on the bull ( n=5 ) sperm viability ( LsmeansA±SEM ) .
Table 2. Effectss of different media ( Milk at 5A°C and 25A°C, Milk with glycerin at 5A°C ) during two different incubation times ( 15 and 60 min ) on the bull ( n=5 ) sperm progressive motility ( LsmeansA±SEM ) .
Tables:
Table 1.
Incubation clip ( min )
Medium
Milk at 5A°C
Milk at 25A°C
Milk with glycerin at 5A°C
15
55A±8.5 aa
85A±8.5 barium
62.8A±5.2 calcium
60
54A±8.5 aa
56A±8.5 Bachelor of Arts
46.4A±5.2 Bachelor of Arts
Bachelor of Arts Valuess with different superior within rows significantly differ ( P & lt ; 0.05 ) .
AB Values with different superior within columns significantly differ ( P & lt ; 0.05 ) .
Table 2.
Incubation clip ( min )
Medium
Milk at 5A°C
Milk at 25A°C
Milk with glycerin at 5A°C
15
29A±9.3 aa
40A±9.3 aa
34.6A±9.3 aa
60
19.4A±9.3 aa
24A±9.3 aa
12.8A±9.3 Bachelor of Arts
Bachelor of Arts Valuess with different superior within rows significantly differ ( P & lt ; 0.05 ) .
AB Values with different superior within columns significantly differ ( P & lt ; 0.05 ) .
Figure Legends:
Fig 1. Bull sperm ( n=4 ) viability after exposure to different osmolarities ( 200, 300 and 400 mOsm/l ) at three different temperatures ( 5, 25 and 39A°C ) for two different incubation times ( 15 and 60 min ) .
Fig 2. Bull sperm ( n=4 ) progressive motility after exposure to different osmolarities ( 200, 300 and 400 mOsm/l ) at three different temperatures ( 5, 25 and 39A°C ) for two different incubation times ( 15 and 60 min ) .
Fig. 1
Fig. 2
