Effect of differential addition of glycerol and pyruvate to extender on cryopreservation of mediterranean buffalo (B.bubalis) spermatozoa

ELSEVIER

EFFECT OF DIFFERENTIAL ADDITION OF GLYCEROL AND PYRUVATE TO EXTENDER ON CRYOPRESERVATION OF MEDITERRANEAN BUFFALO (H.bubalis) SPERMATOZOA A. Fabbrocini, C. Del Sorbo, G. Fasano, and G. Sansone"

Department of General and Environmental Physiology University of Napoli "Federico If' - Italy Received for publication: June 1, 1998 Accepted: Apr;1 6, 2000 ABSTRACT The composition of the extender in which semen is diluted before freezing plays a major role in successful cryopreservation of spermatozoa. Substances of high osmolarity, like glycerol, protect sperm cells during the freezing process and energy-rich compounds, like pyruvate provide extra energy during capacitation and fertilization. Since cryopreservation procedures for Buffalo spermatozoa have not been adequately defined, the aim of the study was to improve the survival rate of buffalo (Hubalus bubalis) spermatozoa after cryopreservation by optimizing the timing for adding glycerol and by enriching the cryoprotectant extender with an energy source substrate. Semen was collected with an artificial vagina from S bulls and the ejaculates were immediately evaluated for motility, forward progressive motility and for viability, pooled and held at room temperature (28°C) for lh. Then aliquots of pooled semen were subjected to dilution and equilibration in triplicate as follows: Experiment 1. Glycerol (3%) in a commercial extender was added to the semen at 28°C and cooled to SoC for 1 h; then extender with 11% glycerol was added before further equilibration (initial glycerol addition; IGA) and the samples held at 5°C for 1, 3 or 5 additional hours (IGA 1, n = 24; IGA 3, n = 24; IGA 5, n = 24) before freezing. Experiment 2. Glycerol (3%) was added and the mixture brought to 5°C as described above. Then extender with 11% glycerol was added (late glycerol addition; LGA) and after equilibration for 1, 3 and 5 h (LGA 1, n= 24; LGA 3, n = 24; LGA 5, n = 24) the samples were frozen. In Experiments 3 and 4 Na pyruvate (1.25 mM) was added to the extender as described for IGA and LGA above (IPA and LPA samples). The effect of addition time (initial vs late) of glycerol and pyruvate was evaluated by measuring sperm motility, progressively forward motility and viability. After freezing-thawing the percentage of motile spermatozoa was significantly higher (O.OOI
Key words: Buffalo, semen, extender, cryoprotectants, energy source

Acknowledgments Research supported by National Research Council of Italy, Project Biotechnology. The authors thank APA (Animal Production Association, Ancona-Italy) for the generous donation of buffalo semen. "Correspondence and reprint request. Theriogenology 54:193-207, 2000 2000 ElsevierScienceInc.

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The composition of the extender in which the semen is diluted before freezing is one of the main factors that influences the success of cryopreservation (7, 11, 29). Extenders containing lipids, sugars and amino acids exert a stabilising effect on the plasma membrane (2, 15, 22) which is needed to maintain the physiologic integrity of the spermatozoa after thawing (8). In addition, extenders should contain compounds with high osmolarity, like glycerol, to protect the sperm cell during freezing (13, 25). In contrast, the presence of glycerol during thawing reduces the fertilizing capacity of cryopreserved semen (1) by inducing osmotic shock (14, 27) or premature capacitation (6, 18). For these reasons, the amount of glycerol and the timing protocol for adding it to the extender are important for success in cryopreservation. For freezing buffalo semen glycerol is commonly added to the extender in concentrations of6 or 7% (20,21) in a lor 2-step dilution protocol, with the second dose being higher than the first. In addition to protecting the spermatozoa from freezing damage, the extender must provide a substrate source of high energy for the spermatozoa during thawing, transport and fertilization (5). Such a substrate is sodium pyruvate (Na pyruvate), an intermediate product of the glycolytic cycle, which is a safe and easy to obtain source of energy for the cells (3).

Our objective was to investigate which equilibration interval from addition of glycerol and pyruvate till freezing would result in best motility and viability of buffalo @ubalus bubalis) spermatozoa after thawing. MATERIALS AND METHODS Semen Collection and Evaluation Semen was collected from 5 buffalo bulls housed at a breeding center of the Italian Breeders Association. An artificial vagina thermoregulated at 42 ± 2°C was used to collect 1-2 ejaculates per bull. Aliquots of each semen sample (1 mL) were taken and immediately evaluated for motility, progressively forward motility and viability. The best ejaculates of each bull were pooled. Additional evaluations were done after each designated cooling interval and after thawing. Motility was estimated using a Makler chamber modified for the Invertoscope Olympus ITM2 (Kontron Instruments, Milano, Italy) equipped with a thermoregulated table maintained at 39°C. Spermatozoa were videotaped and images analyzed for kinetic parameters using a CellSoft CASA system (Cell Soft Europe, Amsterdam, The Netherlands). The percentage of viable sperm cells was determined using the Eosin test according to Rochwerger and Cuaniscu (23). Cooling and Equilibration Protocol Eight collections were made; for each collection the semen from 5 bulls was pooled and all the experimental protocols were tested in triplicate (n = 24 for each experiment protocol) (Figures 1 to 4). Immediately upon collection (Time 0) I-mL aliquots of raw semen were taken for the cooling and equilibration experiments. Raw semen was allowed to cool from 39 to 28°C for 1 h and then subjected to the following treatments.

;1

Figure I. Protocol for studying the effect of adding glycerol to semen at the initial dilution and equilibrating the extended semen for 1,3, and 5h at 5°C before freezing in 0.25 mL straws. The commercial semen extenders Laiciphos A and B contain 3 and II % glycerol, respectively . Semen was evaluated for motility and viability after thawing.


5"

~o

0-

~ Experiment

Procedures

IGA I"

Collect semen Cool from 39 to 28°C for 1 h

Collect semen Cool from 39 to 28 °C for 1 h

IGA3

IGA5

Collect semen Cool from 39 to 28 °C for 1 h

I

o

To I mL semen add 4 mL Laiciphos A cool from 28 to 5°C for 1 h To I mL semen add 4 mL Laiciphos A cool from 28 to 5°C for I h To 1 mL semen add 4 mL Laiciphos A cool from 28 to 5°C for I h

I

I

To 5 mL [semen

+ Laiciphos A] add

freeze

5 rnL Laiciphos B Equilibrate for I h

To 5 mL [semen + Laiciphos A] add 5 rnL Laiciphos B Equilibrate for 1 h

freeze

To 5 mL [semen

+ Laiciphos A] add 5 mL Laiciphos B Equilibrate for 1 h

I 2

freeze

I

II

3

I 4

I 5

I 6

1_ 7

Hour after collection of semen "Initial glycerol addition

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Figure 2. Protocol for studying the effect of adding glycerol to semen after equilibrating for 1,3, and 5h at 5°C. The commercial semen extenders Laiciphos A and B contain 3 and 11% glycerol, respectively. Semen was frozen in 0.25 mL straws and evaluated for motility and viability after thawing.

Experiment

Procedures

LGA I"

Collect semen Cool from 39 to 28°C for 1 h

LGA3

Collect semen Cool from 39 to 28°C for 1 h

LGA5

Collect semen Cool from 39 to 28°C for 1 h

I

o

To 1 mL semen add 4 mL Laiciphos A cool from 28 to 5°C for 1 h To 1 mL semen add4mL Laiciphos A cool from 28 to 5°C for 1 h To 1 mLsemen add 4 mL Laiciphos A cool from 28 to SoC for 1 h

I

1

To 5 mL [semen

+ Laiciphos A] add S mL Laiciphos B Equilibrate for 1 h

freeze

To 5 mL [semen + Laiciphos A] add 5 mL freeze Laiciphos B Equilibrate for 1 h

equilibrate for 2 h

To 5 mL [semen

+ Laiciphos A] add 5 mL Laiciphos B Equilibrate for 1 h equilibrate for 4 h

I

2

I II 3

Hour after collection of semen "Later glycerol addition

I 4

I 5

I

I

67

freeze

~

(l)

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s

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Figure 3. Protocol for studying the effect of 1.25 mM Na pyruvate on semen when added during the initial dilution, before equilibration for 1, 3, and 5h at 5°C . The commercial semen extenders Laiciphos A and B contain 3 and 11% glycerol, respectively. Semen was frozen in 0 .25 mL straws and evaluated for motility and viability after thawing. Experiment

~.

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0-

~

Procedures

IPA I"

Collect semen Cool from 39 to 28°C for 1 h

IPA3

Collect semen Cool from 39 to 28°C for 1 h

IPA 5

Collect semen Cool from 39 to 28°C for 1 h

I

o "Initial pyruvate addition

To 1 mL semen add 4 mL Laiciphos A containing 1.25 mM Na pyruvate cool from 28 to 5°C for 1 h To 1 mL semen add 4 mL Laiciphos A containing 1.25 mM Na pyruvate cool from 28 to 5°C for 1 h To 1 mL semen add 4 mL Laiciphos A containing 1.25 mM Na pyruvate cool from 28 to 5°C for 1 h

11

To 5 mL [semen + Laiciphos A] add freeze 5 mL Laiciphos B containing 1.25 mM Na pyruvate Equilibrate for 1 h To 5 mL [semen

+ Laiciphos A] add 5 mL Laiciphos B containing 1.25 mM Na pyruvate Equilibrate for 1h

equilibrate for 2 h

freeze

To 5 mL [semen + Laiciphos A] add 5 mL Laiciphos B containing 1.25 mM Na pyruvate Equilibrate for 1 h

equilibrate for 4 h

I2

I II

3 Hour after collection of semen

I

4

I

5

~ 6

freeze

7

~

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Figure 4. Protocol for studying the effect of 1.25 mM Na pyruvate on semen when added after equilibration for 1, 3, and 5h at 5°C. The commercial semen extenders Laiciphos A and B contain 3 and 11% glycerol, respectively. Semen was frozen in 0.25 mL straws and evaluated for motility and viability after thawing. Experiment

Procedures

LPA I"

Collect semen Cool from 39 to 28°C for I h

LPA3

Collect semen Cool from 39 to 28°C for 1 h

LPA5

Collect semen Cool from 39 to 28°C for I h

I o

To 1 mL semen add 4 mL Laiciphos A without Na pyruvate cool from 28 to 5°C for 1 h To 1 mL semen add 4 mL Laiciphos A without Na pyruvate cool from 28 to 5°C for 1 h

To 5 mL [semen + Laiciphos A] add 5 mL Laiciphos B freeze containing 2.5 mM Napyruvate Equilibrate for 1 h To 5 mL [semen + Laiciphos A] add 5 mL freeze Laiciphos B containing 2.5 mMNapyruvate Equilibrate for lh

equilibrate for 2 h

To 1 mL semen add4mL

To 5 mL [semen + Laiciphos A] add 5 mL Laiciphos B containing 2.5 mM Na pyruvate Equilibrate for 1 h

Laiciphos A

without Na pyruvate cool from 28 to 5°C for 1 h

'1

equilibrate for 4 h 1

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2

3

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Hour after collection of semen "Later pyruvate addition

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Initial Glycerol Addition (IGA). We used Laiciphos®, a commercial extender (IMV-Italia, Piacenza, Italy) that contains egg yolk and was prepared with glycerol (Sigma-Aldrich, Milano, Italy) in 2 concentrations (Laiciphos A, 3% and Laiciphos B, 11%) To 1 mL of the undiluted, cooled semen were added 4 mL Laiciphos A and the samples cooled from 28 to 5°C for 1 h. At this time to the 5 mL aliquot of the diluted semen were added 5 mL of Laiciphos B resulting in a final glycerol concentration of 6.7% (0.128M). The samples were then assigned to a pre-freezing equlibration time of I, 3 and 5 h, designated as IGA 1, IGA 3 and IGA 5, respectively, as shown in Figure 1. . Late GlycerQl Addition (LGA). In this experiment the second glycerol addition was after the equilibration. After the initial cooling, dilution with Laiciphos A and equilibration of the semen to 5°C as described above, 5 mL ofLaiciphos B were added to the 5 mL of diluted semen and the samples equilibrated for 1 h at 5°C and frozen (LGA 1). The second and third sets of samples (LGA 3 and LGA 5) were equilibrated for 2 and 4 h, respectively, then 5 mL of Laiciphos B were added and the samples equilibrated for an additional 1 h, for a total of 3 and 5 h and frozen (Figure 2). Injtial Pyruvate Addition (IFA). In this experiment both Laiciphos A and B were enriched with 1.25 mM of Na pyruvate (Sigma-Aldrich, Milano, Italy), a concentration commonly added to in vitro fertilization media (1). The remainder of the protocol was as described above for the late addition of glycerol (Figure 3). In this experiment, we used as control, besides fresh semen, the LGA 5 protocol, which gave the best results in the first and second experiments, to verify the real improvement of the protocol after adding Na pyruvate. Late Pyruvate Addition (LPA). In the final experiment Laiciphos A was without Na pyruvate but Laiciphos B contained 2.5 mM when added to semen after the 0-, 2-, and 4equilibration intervals each followed by freezing 1 h later. The final Na pyruvate concentration was 1.25 mM. The remainder of the protocol was as described above for the late addition of glycerol (Figure 4). Also in this experiment, we used as control, besides fresh semen, the LGA 5 protocol which gave the best results in the first and second experiments, to verify the real improvement of the protocol after adding Na pyruvate. Freezing and Thawing Protocol At the end of the cooling and equilibration procedures, the semen was packaged into 0.25 mL straws and frozen by a stepwise protocol as follows: cooled from +5 to -15°C at 40°C/min, pause for 7min, cooled from -15 to _80°C at 240°C/min, pause for 15min, cooled from -80 to 100°C at 300°C/min, pause for 15min and finally immersed in liquid nitrogen and stored. The straws were thawed by immersion in a waterbath at 39°C for 30 seconds. Statistical analysis Mean percentages of motile and progressively motile spermatozoa observed in the treated semen were compared to the untreated (fresh) semen and differences were at first evaluated by

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ANOVA, and successively by Student's t test, with P < 0.05 being considered as a significant differences. For the 8 collections made each experiment protocol was tested in triplicate (n = 24 for each experiment protocol) . RESULTS Glycerol Addition protocol (IGA and LGA) The percentage of live cells was always more than 90%. During the cooling phase there were no significant differences in the percentages of both motile and progressively forward motile spermatozoa compared to fresh semen, .for both the experiment protocols. On thawing, motility (Figure 5) was significantly lower than that of fresh semen (0.001
T

o Collection

80

60 140 20 • 0 Fresh

1hour

3hours

5hours

Figure 5. Mean (± SD vertical bars; n = 24) percentages of motile spermatozoa in fresh semen and thawed semen frozen after addition of glycerol-containing extender at different times before freezing. Glycerol was added during the initial dilution and the extended semen equilibrated for I, 3 and 5 h at 5°C before freezing (IGA). Glycerol was added after equilibration for 0, 2 and 4 h and after lh the semen was frozen (LGA). Fresh semen was significantly better than treated semen (O.OOI
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The analysis of progressively forward motility data by ANOVA shows that equilibration time has a significant effect (P<0.001) greater than that of the addition protocol (p=0 .007). In fact, for both the two glycerol addition procedures the lhr equilibrated samples are significantly lower than the 3hr and 5hr ones (P<0.001), while no significant differences can be found among the two protocols . 100

o Total motile sperm

• Forward sperm 80

60 40 20

o fresh

lOA Ihour

LOA Ihour

lOA 3hours

LOA

lOA

LOA

3hours

Shours

Shours

Figure 6. Mean (± SD vertical bars; n = 24) percentages of progressively forward motile spermatozoa in fresh semen and thawed semen frozen after addition of glycerolcontaining extender at different times before freezing. Glycerol was added during the initial dilution and the extended semen equilibrated for I, 3 and 5 h at 5°C before freezing (IGA) . Glycerol was added after equilibration for 0, 2 and 4h and after Ih the semen was frozen (LGA). Fresh semen was significantly better than treated semen (P
202

TheriogenoJogy

100

~

o Collection

• Control

DLPA

80

60

o

I ,-_ L -l -_ _-r-'

Fres h

LGA Shours

Iho ur

3hours

Shours

Figure 7. Mean (± SD vertical bars; n "" 24) percentages of motile spermatozoa in fresh semen and semen after addition of Na pyruvate enriched extender at different times before freezing. For IPA samples both Laiciphos A and B contained 1.25mM Na pyruvate, while for LPA samples only Laiciphos B contained 2.5mM Na pyruvate. Laiciphos B and glycerol were added according to the LGA procedure. LGA 5 was used as control to verify the real improvement of the protocol after adding Na pyruvate. One hand 3h equilibrated samples were significantly better than the control sample (002
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TheriogenoJogy 100

OTOlal motile sperm

• Forward sperm

80 60 40 20

o Fresh

Control

IPA

J hour

LPA I hour

IPA 3hours

LPA 3hours

IPA 5hours

LPA 5ho urs

Figure 8. Mean (± SD vertical bars; n = 24) percentages of progressively forward motile spermatozoa in fresh semen and semen after addition of Na pyruvate enriched extender at different times before freezing. For IPA samples both Laiciphos A and B contained 1.25mM Na pyruvate, while for LPA samples only Laiciphos B contained 2.5mM Na pyruvate . Laiciphos B and glycerol were added according to the LGA procedure. LGA 5 was used as control to verify the real improvement of the protocol after adding Na pyruvate . In the Ihr and 3hr IPA equilibrated samples and Ihr and 5hr LPA equilibrated samples progressive motility was significantly lower than the LGA 5 sample (O .OOl
On thawing, no significant differences in motility (Figure 9), were observed either versus fresh semen and control, except for IPA 3, which was significantly lower (P
All the samples showed a lower progressively forward motility (Figure 10) with respect to fresh semen and control (P
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100

~

o Collection

Control

. LPA

. IPA

80 60

40

1

20 -+--

0 LGASh oup.;

Fresh

---j

Shours

Ihour

Figure 9. Mean (± SD vertical bars; n = 24) percentages of motile spermatozoa in fresh semen and thawed semen frozen after addition ofNa pyruvate enriched extender at different times before freezing. For IPA samples both Laiciphos A and B contained 1.25mM Na pyruvate, while for LPA samples only Laiciphos B contained 2.5mM Na pyruvate. Laiciphos B and glycerol were added according to the LGA procedure. LGA 5 was used as control to verify the real improvement of the protocol after adding Na pyruvate. Fresh semen was significantly better than treated semen (O.OOl
• Forward sperm

Fresh

Control

IPA Ihour

LPA Ihour

o Total motile sperm

II'A 3hoors

LI'A Jhours

IPA Shours

LPA Shours

Figure 10. Mean (± SD vertical bars; n = 24) percentages of progressively forward motile spermatozoa in fresh semen and semen after addition of Na pyruvate enriched extender at different times before freezing. For IPA samples both Laiciphos A and B contained 1.2SmM Na pyruvate, while for LPA samples only Laiciphos B contained 2.5mM Na pyruvate. Laiciphos B and glycerol were added according to the LGA procedure. LGA 5 was used as control to verify the real improvement of the protocol after adding Na pyruvate. Fresh semen was significantly better than treated semen (O.OOI
Theriogenology

205 DISCUSSION

Glycerol at various concentrations has been used as a universal cryoprotective agent for freezing spermatozoa. It modifies the crystal formation in the medium by preventing the electrolyte concentration from rising above a harmful level (20, 26, 28). But besides its cryoprotective role, it has also been reported to produce a deleterious effect on the fertilizing potential of spermatozoa (19) . Hammerstedt (17) remarked on the various potential actions of glycerol, not least of which were the osmotic stress associated with the introduction and removal of molar concentrations from the cell interior, the modification of the plasma-membrane by its ability to insert itself among the phospholipids, and its potential to become involved in intermediary metabolic pathways. Glycerol is commonly added to the extender for freezing buffalo semen in concentrations from 6 to 7%, despite many unsuccessful attempts to reduce the amount or to substitute it with other cryoprotective substances (20, 21). It may be added to the semen in one amount or in two steps, the second having a higher cryoproteetant concentration than the first. Del Sorbo (10) tested monophasic and biphasic TRIS-yolk- based extenders and observed that biphasic extenders give better results with long equilibration times before freezing, while monophasic extenders require shorter equilibration times. The results obtained in the first two experiments, in which we aimed to define the best time for adding glycerol, showed that with the LGA procedure both total motility and progressively forward motility increased as the equilibration time increased, while with the IGA protocol they both decreased when the equilibration time increased ; these results suggest that a cooling procedure with a slow temperature decrease and a long acclimatization phase in presence ofa small amount of glycerol, improves dehydration of the cell, thus limiting cell damage. The first series of experiments also shows that even if motility decreases during equilibration time and after thawing in comparison with ejaculated semen, there are always more than 90% live spermatozoa, so that in thawed samples there are a lot of alive but immotile spermatozoa. These findings support our previous observation that after incubation of thawed buffalo semen at 39°C there was a rapid decrease in motility, but no increase in the percentage of dead spermatozoa (9). On the basis of these results , we supposed that all the immotile cells needed a high energy source to help them overcome the stress of the freezing/thawing procedure. Sodium pyruvate is generally added to the culture media to enhance sperm motility during the procedures for the selection of better quality sperm such as "swim up" (3, 4, 12, 16,24). In the second series of experiments, results showed that the enrichment of the extender with Na-pyruvate enhanced sperm motility during the cooling time ; the percentages of motile spermatozoa after Ih and 3h of cooling were in fact significantly better than those obtained with the LGA5 procedure, which gave the best results in the first experiment and was used as the control in the second. On the contrary , no significant differences were found for the two different addition procedures. The difference among the two protocols were be observed on thawing : the LPA procedure seemed to preserve a higher percentage of motile spermatozoa than the 1PA procedure, especially for longer equilibration times, when motility was significantly better than theLGA5 .

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Therefore, Na pyruvate can be used as an energy source to help the spermatozoa to overcome stress due to freezing/thawing only if added a short time before freezing (so that the most effective use will come about on thawing and not during cooling and equilibration); in particular, its effects are clearly seen after long equilibration times, during which, in all probability, the spermatozoa face a greater energy expenditure. In conclusion, for freezing buffalo spermatozoa we suggest adding the extender to the semen in two steps; the second aliquot of extender, containing sodium pyruvate and the greater amount of glycerol, should be added only Ihr before freezing, and an equilibration time of no longer than 5h should be applied. REFERENCES I. Abdelhakeam AA, Graham EF, Vazquez IA, Chaloner KM. Studies on the absence of glycerol in unfrozen and frozen ram semen. Cryobiology 1991;28: 43-49 . 2. Anchordoguy TJ, Rudolph AS, Carpenter JF, Crowe JH. Models of interaction of cryoprotectants with membrane phospholipids during freezing. Cryobiology 1987;24: 324331. 3. Bavister BD, Yanagimachi R. The effect of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol Reprod 1977;28: 235247. 4. Chan YM, Abuzeid MI, Malcomnson JH, Sasy MA Selection of human spermatozoa by a hyperosmotic two-layer Percoll gradient. Fertil Steril 1994;61(Suppl 6): 1097-1102. 5. Chen Y, Foote RH, Brockett CC. Effect of sucrose, trehalose, hypotaurine, taurine and blood serum on survival of frozen bull sperm. Cryobiology 1993;30: 423-431. 6. Cormier N, Sirard MA, Bailey JL. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J Andro11997;18 (SuppI4): 461-468. 7. De Leeuw FE, De Leew AM, Den Daas JHG, Colenbrander B, Verkleij AI. Effects of various cryoprotective agents and membrane-stabilizing compounds on bull sperm membrane integrity after cooling and freezing. Cryobiology 1993;30: 32-44. 8. De Leeuw FE, Hsiao-Ching C, Colenbrander B, Verkleij AJ. Cold-induced ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology 1990;27: 171-183. 9. Del Sorbo C, Fabbrocini A, Grasso F, Nastri MJF, Talevi R, Sansone G, Matassino D. Studio preliminare sulla qualita di spermatozoi bufalini (B.bubalis) allo scongelamento in diversi mezzi di diluizione. Prod Anim 1992;5,(III Serie): 61-68. 10. Del Sorbo C, Fasano G, Fabbrocini A, Nastri MJF, Sansone G. Studio preliminare su effetti di vari agenti crioprotettivi sulla motilita di spermatozoi bufalini (B.bubalis) allo scongelamento. Proc of 6th Meeting Nazionale "Studio della efficienza riproduttiva degli animali di interesse zootecnico" 1994; 1:63-67. I I. Dhami AJ, Jani VR, Mohan G, Sahni KL. Effect of extenders and additives on freezability, post-thaw thermoresistance and fertility of frozen Murrah buffalo semen under tropical climate. Buffalo J 1994;1: 35-45. 12. Drobnis EZ, Zhong CQ, Overstreet TW. Separation of cryopreserved human semen using Sephadex columns, washing, orPercoll gradient. J Andro11991;12 (SuppI3): 201-208. 13. Fiser PS, Fairfull RW. Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar speramtozoa frozen in 0.5 ml straws. Molec Reprod Dev 1990;25: 123-129. 14. Fiser PS, Fairfull RW. The effect of glycerol-related osmotic changes on post-thaw motility and acrosomal integrity ofram spermatozoa . Cryobiology 1989;26: 64-69.

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15. Goodrich RP, Baldeschweiler JD. The cryoprotective action of synthetic glycolipids. Cryobiology 1991;28 : 327-334. 16. Grant SA, Long SE, Parkinson TI. Fertilizability and structural properties of boar spermatozoa prepared by Percoll gradient centrifugation. J Reprod 1994; I00 : 477-483 . 17. Hammerstedt RH, Graham JK. Cryopreservation of poultry semen: the enigma of glycerol Cryobiology 1992; 29 : 26-38 . 18. Holt WV , Medrano A. Assessment ofboar sperm function in relation to freezing and storage. J Reprod Fertil Suppl 1997;52: 213·222. 19. Kumar S, Sahni KL, Benjamin BN , Mohan G. Effect of various levels of yolk on deep freezing and storage of buffalo semen in different diluters without adding glycerol. Buffalo J 1993;1: 79-85 . 20. Kumar S, Sahni KL, Mohan G. Effect of different levels of glycerol and yolk on freezing and storage of buffalo semen in milk, tris and sodium citrate buffers . Buffalo J 1992;2: 151156. 21. Kumar S, Sahni KL, Mohan G. Effect of different sugars as sole cryoprotectant in freezing of buffalo semen. Buffalo J 1992; 13: 305·309. 22. Parkinson TJ, Whitefield CH. Optimisation of freezing conditions for bovine spermatozoa. Theriogenology 1987;27 : 781-798. 23. Rochwerger L, Cuaniscu PS . Redistribution of a rat sperm epididymal glycoprotein after in vivo and in vitro capacitation. Mol Reprod Dev 1992;31: 34-41. 24. Smith SMD , Hodis SRN , Scott LMS . Use of postseparation sperm parameters to determine the method of choice for sperm preparation for assisted reproductive technology. Fertil Steril 1995;63 (SuppI3) : 591- 597. th 25. Vale WG . News on reproductive biotechnology in males. Proc 5 World Buffalo Congr 1997 ;1: 103-12 3. 26. Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function . Reprod Fertil Dev 1995;7: 871-891. 27. Watson PF , Duncan AE . Effect of salt concentration and unfrozen water fraction on the viability of slowly frozen ram spermatozoa. Cryobiology 1988;25: 131-142. 28. Woelders H. Fundamentals and recent development in cryopreservation of bull and boar semen . Vet Q 1997;19 (SuppI3): 135-138 . 29. Woelders H, Matthijis A, Engel B. Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing Cryobiology 1997 ;35 (Suppl 2): 93-105 .