Frozen storage of semen

Ž . In studies of different egg yolk 1, 2.5, 5, 10, 20 concentrations in Ž . tris-based diluent, the low levels 1 and 2.5 showed the best sperm survival rates Ž . during 72 h storage at 58C Sahni and Mohan, 1990 . There was no improvement in viability of spermatozoa after 72 h stored at 58C when 5, 10 and 20 egg yolk was included in milk- or tris-based extenders, but in the latter diluent the spermatozoa Ž . survived better in the presence of 10 or 20 than 5 egg yolk Kumar et al., 1993b . Published reliable reports on fertility of liquid stored buffalo semen seem not to be Ž . available editorial comment .

5. Frozen storage of semen

5.1. Diluents An important factor influencing frozen storage of semen is the composition of the medium used for dilution of semen before freezing. The diluent is usually composed of a buffer medium to which cryoprotectants and other substances are added, which protect the spermatozoa during freeze–thawing. Antibiotics are commonly added to the exten- der. Ž . In studies of Dhami et al. 1994 , tris–fructose–egg yolk, citrate–egg yolk and lactose–egg yolk diluents did not differ in their ability to protect spermatozoa against freeze–thawing damages. However, the tris-based diluent yielded higher post-thaw Ž . Fig. 2. Tris–yolk-based extender: sperm vision is not very clear Obj. 20= . Ž . motility compared with the other two media 54.1, 46.5 and 41.6, respectively . Ž . Other workers Raizada et al., 1990; Chinnaiya and Ganguli, 1990 also found no difference in post-thaw motility of spermatozoa frozen in tris- or citrate-based extenders. Both tris- and lactose-based media were superior to the milk diluent as determined by Ž . post-thaw motility Akhtar et al., 1990 . In other in vitro comparisons of diluents, based on post-thaw motility of spermatozoa, Ž . milk was better than tris Galli et al., 1993 , or both these media performed better than Ž . the citrate-based extender Kumar et al., 1992a . However, in a subsequent test, the performance of citrate medium was similar to that of commercial milk-based diluent, Ž and was also found sufficiently clear for computerised analysis of motility see Figs. 2 . Ž . and 3 Del Sorbo et al., 1995a . wŽ . . Tes Hydroxymethyl aminoethan is another buffer used for freezing buffalo semen, Ž and proved to be of equal value to tris- and glycine-based diluents Oba et al., 1994; . Chachur et al., 1997 . Ž . Galli et al. 1993 suggested that the differences regarding in vitro performance of the various extenders, which may not emerge in tests with bovine semen, indicate that buffalo spermatozoa are more prone to freezing stress. The poor freezability may be correlated to the low membrane phospholipid content and to its loss during freeze–thaw- ing. 5.1.1. Cryoprotectants Both egg yolk and glycerol are used in combination as cryoprotectants. The lecithin and lipoprotein contents in egg yolk contribute to the preservation of the lipoprotein Ž . sheath of the sperm cell Kumar et al., 1992a . However, in addition to its protective Ž . Fig. 3. Citrate–yolk-based extender: sperm vision is better than in the tris-based medium Obj. 20= . action against cold shock, the egg yolk also stimulates the enzyme system of spermato- zoa. This results in deamination of certain specific amino acids normally present in the dialysable fraction of egg yolk and yields hydrogen peroxide, which is toxic to Ž . spermatozoa during storage under aerobic conditions Sahni and Mohan, 1990 . For this reason, the egg yolk may be dialysed before addition to the extender. Little attention has been paid to the level of egg yolk necessary for freezing buffalo semen, and the majority of workers are using concentrations around 20. It should be noted, however, that as the yolk concentration is increased in the diluent, the pH of the medium decreases and tends towards the acidic side. This may also be the reason for the depressing effect of higher amounts of yolk on motility of thawed spermatozoa. The toxic effect of egg yolk may be Ž . combined with toxicity of dead spermatozoa Shannon, 1972 . Ž . Sahni and Mohan 1990 examined various levels of egg yolk in a tris–glycerol-based freezing diluent and found that egg yolk beyond 5 did not show any significant improvement in post-thaw motility. This indicated the scope of reducing the yolk level from 20 to 5 without adversely affecting the freezability of buffalo semen. In the Ž . absence of egg yolk, none of the diluents used tris-, milk- and citrate-based gave protection to spermatozoa during freeze–thawing. After addition of egg yolk, post-thaw motility improved significantly, but there was no further increase for concentrations Ž . higher than 5 in the three buffers Kumar et al., 1993b . Glycerol is generally added to the extender for freezing buffalo semen in concentra- tions from 6 to 7, but attempts have been made to reduce the amount, or substitute it with other cryoprotective substances. Reduction of glycerol to 3 or 2, and concentra- tions above 7 decreased the post-thaw motility of spermatozoa in the diluents tested Ž . Kumar et al., 1992a; Ramakrishnan and Ariff, 1994; Nastri et al., 1994 . The cryoprotective effects of six sugars — glucose, xylose, raffinose, fructose, Ž . sucrose and cheeni a high molecular weight sugar containing 99.5 sucrose each at Ž . 1, 1.5 and 2 concentrations, were examined by Kumar et al. 1992b . They found that the protective effect of sugars depended on the type of diluent used for freezing. In milk diluent, fructose and sucrose gave the best protection, as judged by post-thaw motility results, due probably to the synergy action of lactose present in milk. Some protection to spermatozoa was given by the sugars at 2 concentration in tris diluent, and none in citrate-based medium. The high molecular weight sugars may play a cryoprotective role by altering the permeability of the cell membrane and by maintaining the electrolyte balance. However, the best post-thaw motilities were never higher than 30 when sugar was used as the sole cryoprotectant. The use of extenders of vegetable origin for the preservation of live cells has long been known. Some vegetable derivatives, like coconut water and tomato juice, seem to have properties that protect bovine and caprine spermatozoa. Coconut water has been shown to be an alternative for storage of bovine semen, as the phytohormones it contains Ž protect the spermatozoa against the phospholipase A enzyme in the ejaculate Nunes, . Ž . Ž . 1993 . Nunes et al. 1996 reported that indole-3-acetic acid IAA in coconut water appeared to be the main protective substance for spermatozoa. The suitability of coconut Ž . water-based diluent for processing buffalo semen was shown by Vale et al. 1997 . The coconut water obtained from fresh fruit must be filtered and, at its final state before use for dilution, should have an osmolarity of 320 mosM and pH 6.8. 5.1.2. AdditiÕes to freezing diluents Many attempts have been made to improve the basic buffers developed in the early 1950s by inclusion of additives such as vitamins, amino acids, chelating agents, enzymes, metabolic stimulants and others. Seminal plasma of domestic animals contains mainly water soluble vitamins C and B. The role of vitamins A, D and E in the viability and fertilising ability of spermatozoa is Ž . Ž . not clear. Kolev 1997 examined the effects of vitamins A 100 and 200 IUrml , D Ž . Ž . 200 and 400 IUrml and E 0.3 and 0.5 mgrml added to the freezing diluent for buffalo semen. The best post-thaw motility was obtained after addition of vitamin E at Ž . 0.3 mgrml dose. Satisfactory motility was also obtained with vitamin A 100 IUrml . Thus, some of the vitamins may have a positive effect on post-thaw viability of buffalo spermatozoa. Ž . Ž . Dhami and Sahni 1993 and Dhami et al. 1994 examined the effects of raffinose Ž . Ž . Ž . 1 , L -cysteine 0.1 and EDTA 0.1 in the generally used freezing media for Ž . buffalo semen tris-, milk- citrate-based diluents . The beneficial effects of raffinose on post-thaw motility and the fertility rate were observed only in the citrate buffer which did not contain other sugar. L -cysteine and EDTA improved the fertility rate, compared to raffinose or the control extenders without additive. This was attributed to significantly Ž . better progressive motility of spermatozoa when chelating agents cysteine and EDTA were present in the diluent. Citric-whey extender supplemented with 0.1 and 0.2 Ž . cysteine increased post-thaw sperm motility, particularly at the higher 0.2 concentra- Ž . tion Singh et al., 1990 . Sodium pyruvate, an energy source that can be readily used by the cells, had a beneficial effect on post-thaw sperm survival only when added 1 h before freezing to Ž . citrate–yolk–glycerol diluent Del Sorbo et al., 1995b . Caffeine was found to stimulate Ž . motility of buffalo spermatozoa. El-Menoufy et al., 1985 . 5.1.3. Antibiotics Ž . Ž . Penicillin 1000 IUrml and streptomycin sulphate 1.0 mgrml alone or in combina- Ž . tion are commonly added to freezing diluents. Aleem et al. 1990 examined the microorganisms present in buffalo bull semen, and their sensitivity to commonly available antibiotics. They found that a combination of penicillin and neomycin was more effective than the combination of penicillin and streptomycin currently used. Table 4 describes the composition of some diluents recommended for freezing buffalo semen. 5.2. Processing of semen 5.2.1. Transfer of semen samples The breeding farms are often far from laboratories and there may be a delay before semen samples are processed. No changes have been observed in motility, morphology or freezability, of spermatozoa, if the semen was processed within 1 h of collection Ž . Ž . Fabbrocini et al., 1995 . Vale et al. 1991 recommended to keep the ejaculate in its own plasma for 10–15 min, although semen of some buffaloes may show agglutination. In this case, addition of the diluent immediately after collection could prevent the semen Table 4 Diluents for freezing buffalo semen Ž . Ž . A Tris-based diluent recommended by Ramakrishnan and Ariff 1994 1st step 2nd step Egg yolk 20 20 Tris 3.02 3.02 Citric acid 1.67 1.67 Ž . D y Fructose 1.25 1.25 Glycerol 4 12 Penicillin 1000 IUrml 1000 IUrml Streptomycin 1.0 mgrml 1.0 mgrml Bidistilled water to 1000 ml to 1000 ml Ž . Ž . B Egg yolk–galactose–cysteine diluent recommended by Del Sorbo et al. 1995b 1st step 2nd step Egg yolk 20 20 Ž . D y galactose 0.5 0.5 Cysteine 6.4 mM 6.4 mM Caffeine 7 mM 7 mM Vitamin C 0.7 mM 0.7 mM Na-pyruvate – 2.5 mM Glycerol 0.3 12.3 Penicillin G 60 mgrl 60 mgrl Citraterhepes buffer 0.2 M — pH 6.8 to 1000 ml to 1000 ml Ž . Ž . C Egg yolk–tes–tris–fructose–milk diluent recommended by Vale 1997 Stock solution I 36.5 ml Stock solution II 36.5 ml Glycerol 7.0 ml Egg yolk 7.0 ml Ž . Adjust pH to 6.8–7.0 with NaOH 10 Stock solution I Tes 48.3 g Tris 11.6 g Ž . D y Fructose 2.0 g Bidistilled water 1000 ml 6 Penicillin G 1=10 IU Streptomycin sulphate 2.0 g Stock solution II Skim milk 110.0 g Bidistilled water 1000 ml Ž . Heat at 928C for 5 min Ž . Ž . D Coconut-water diluent according to Vale et al. 1997 Stock solution II 93.0 ml Glycerol 7.0 ml Penicillin potassium G 1000 IUrml Streptomycin sulphate 2.0 gr100 ml Ž . Adjust pH to 6.8–7.0 with NaOH 10 Ž . Table 4 continued Ž . Ž . D Coconut-water diluent according to Vale et al. 1997 1st step 2nd step Stock solution I Coconut water 50.0 ml Bidistillated water 25.0 ml Sodium citrate 5 25.0 ml q.s.p. 25.0 ml Stock solution II Stock solution I 90.0 ml Egg yolk 10.0 ml q.s.p. 100.0 ml from irreversibly agglutinating and also maintain the motility of spermatozoa. If some hours are needed before the beginning of semen processing, dilution of semen immedi- ately after collection with the freezing medium and subsequent storage at 58C is Ž . suggested Talevi et al., 1994 . These workers found that the percentage of motile spermatozoa remained consistent for up to 6 h at 58C in a diluent containing 0.3 glycerol. 5.2.2. Methods of dilution The semen is diluted in one or two steps to a final concentration of about 100–150 = 10 6 spermrml. When the two-step method is adapted, the second diluent portion has a higher cryoprotectant concentration than the first portion. Del Sorbo et al. Ž . 1994 examined both methods using tris–egg yolk-based extenders, and found that the Ž . two-step method gave better results with long 6 h equilibration, while the extenders Ž . used for one-step dilution required shorter 2–4 h equilibration time before freezing. Addition of glycerol to semen, using a commercial milk extender and two variants, was Ž . examined by Fabbrocini et al. 1995 . In the first variant, the diluent used for 1st and 2nd step dilution contained 3 and 11 glycerol, respectively, and in the second variant, the glycerol concentrations were 0.3 and 14.3 in the diluent for 1st and 2nd step dilution. The second dilution was done 1 or 6 h before freezing. The only significant improvement in motility was observed when glycerol was added 1 h before freezing. Thus, it seems that addition of glycerol 1 h before freezing is the best method when a two-step dilution is adopted. Two-step dilution with Na–pyruvate was proposed Ž . also by Del Sorbo et al. 1995b who performed the second dilution also 1 h before freezing. 5.3. Cooling of semen Ž . Talevi et al. 1994 examined slow and a rapid cooling procedures: in the first case, Ž . the semen having an initial temperature of 288C reached a temperature of 58C in 1 h, in the second case in 15 min. There was no significant difference in post-thaw motility of spermatozoa cooled rapidly or slowly. Similar cooling rates were also used by Singh et Ž . Ž . al. 1990 and Sahni and Mohan 1990 . Slow cooling procedures were tested by Ž . Ramakrishnan and Ariff 1994 , who cooled the semen to 58C in 45, 65 and 90 min, and Ž . observed no significant difference in post-thaw motility. Dhami and Sahni 1994 cooled the semen from 308C to 58C in 60 and 120 min, and found the latter more advantageous. Ž . In conclusion, a slow cooling procedure 0.2–0.48Crmin is recommended during pre-freezing processing of buffalo semen. 5.3.1. Equilibration of semen There was no agreement among investigators regarding the duration of equilibration. Ž . Ž Some suggested short 2–4 h periods Singh et al., 1990; Dhami and Sahni, 1994; Del . Ž . Ž Sorbo et al., 1995b , while others recommended longer about 6 h equilibration Rao et al., 1990; Chinnaiya and Ganguli, 1990; Dhami and Kodagali, 1990; Haranath et al., . 1990; Talevi et al., 1994 . It is a general belief that buffalo semen should stand at 58C for not less than 2 h and no longer than 6 h before freezing. 5.4. Freezing of semen Ž . After equilibration, the semen packaged in mini straws 0.25 ml is frozen in liquid nitrogen vapour. Mini straws are generally used due to their cost effectiveness and Ž . saving of storage space. Haranath et al. 1990 found an improvement in conception rate Ž . for semen frozen in mini straws as compared to that frozen in medium straws 0.5 ml Ž . 52.7 vs. 50.4 . Freezing in liquid nitrogen vapour is practical and can be done by using a simple isotherm box. The straws are suspended in horizontal position 1–4 cm above liquid nitrogen for 10–20 min, after which they are immersed into liquid nitrogen at y1968C. Ž . Ž . Del Sorbo et al. 1995b examined two freezing procedures: 1 a ‘‘step-wise curve’’, Ž . using a freezing tray Taylor Wharton , on which the straws were exposed to tempera- Ž . Ž . Ž . Ž . tures of q58C 4 min , y158C 7 min , y808C 15 min and y1308C 15 min in Ž . Ž . liquid nitrogen vapour, then immersed into liquid nitrogen see Fig. 4 ; and 2 a ‘‘continuous curve’’ that involved decreases of temperature from q58C to y308C at a speed of 208Crmin, from y308C to y1008C at a speed of 158Crmin, then standing for Ž . Fig. 4. ‘‘Step-wise’’ freezing curve according to Del Sorbo et al. 1995b . Ž . Fig. 5. ‘‘Continuous’’ freezing curve according to Del Sorbo et al. 1995b . Ž . 5 min at y1008C, before immersion into liquid nitrogen see Fig. 5 . The post-thaw recovery rates were better for ‘‘step-wise’’ than for continuous freezing curve. In the Ž . continuous freezing curve of Vale 1997 , the rates of decrease in temperature are Ž . 188Crmin from q48C to y408C and 88Crmin from y408C to y1408C see Fig. 6 . Programmable freezers may be used to monitor the above curves. 5.5. Thawing of semen To avoid recrystallization, rapid warming rates are commonly used. Dhami et al. Ž . Ž . 1994 and Vale 1997 suggested thawing of straws in a water bath at 408C for 30 s. Ž . Ž . Somewhat slower thawing rates were used by Kumar et al. 1993b 378C for 30 s , Ž . Ž . Ž . Ž Ramakrishnan and Ariff 1994 358C for 30 s and Fabbrocini et al. 1995 398C for 30 . Ž . s . El-Amrawi 1997 examined the effects of thawing time and of post-thaw tempera- Ž . ture on the quality of buffalo semen. Medium straws 0.5 ml were thawed in a water bath at 358C for 12 s or 1 min, then transferred to a water bath at 108C, 208C or 308C for 1 min, and again in a water bath at 408C for 1 min. The best post-thaw motility rate Ž . 50.8 was obtained after thawing at 358C for 1 min and subsequent transfer of straws to a post-thaw temperature of 108C. The thawing time had an effect on the internal temperature of the straws: straws thawed for 12 s had an internal temperature of y48C, while those thawed for 1 min reached an internal temperature of 368C, which also Ž . Fig. 6. ‘‘Continuous’’ freezing curve according to Vale et al. 1997 . affected the acrosome integrity. Although some workers state that the quality of buffalo semen frozen in straws can be improved by using a longer thawing time, Ziada et al. Ž . 1992 found no difference between thawing at 358C for 30 s and at 508C for 15 s. 5.5.1. Characteristics of thawed spermatozoa Several parameters are used to evaluate the morphological and physiological state of spermatozoa after freeze–thawing, particularly motility, forward motility and membrane Ž . integrity monitored by lectins and sperm–oocyte interaction. Fabbrocini et al. 1996 examined the integrity of the plasma membrane of thawed spermatozoa using MPA-FITC lectin that binds to lectin-similar receptors on the cell surface. Changes in acrosome membrane that occur during the freeze–thawing procedure may prevent the lectin from binding to the surface sugars, and this could be considered as an evaluation parameter of non-lethal damage to the cell. Another method for evaluation of frozen–thawed semen is the analysis of enzymes Ž . Ž . which may be related to fertility Table 5 . Akhtar et al. 1990 found a significant increase in hyaluronidase activity in frozen–thawed semen using the Rhodes method Ž . Ž . Rhodes et al., 1971 . The activities of aspartate aminotransferase AAT , hyaluronidase Ž . Ž . Ž . aminotransferase HAT , lactic dehydrogenase LDH Bhosrekar et al., 1994 , KPH Ž . Ž . Ž . Kind and King, 1954 and acid phosphatase APH King and Jaggatheesan, 1959 were Ž . measured by Bhosrekar et al. 1994 . They found that the levels of all enzymes in extra-cellular medium increased significantly after freeze–thawing, which indicates leakage of enzymes from sperm cells. The release of HAT and AAT showed a highly significant negative correlation with motility and acrosomal integrity, while LDH, APH and KPH had a positive relation with motility and acrosomal integrity. The transami- nases in spermatozoa are intrinsically associated with their metabolic activity and Ž . function as a reservoir of energy. Kaker and Anand 1984 stressed the importance of levels of GOTrGPT enzymes in the seminal plasma as an indicator of the quality of frozen–thawed semen, since the GOTrGPT release is influenced by factors such as cold Ž . shock, glycerol concentration, cooling and freezing rates. Dhami and Kodagali 1990 Ž . and Dhami and Sahni 1994 utilised the GOTrGTP, AKP, ACP and LDH levels to assess the quality of spermatozoa after freeze–thawing procedures. Fertility rates showed a high negative correlation with the release of all five enzymes, which indicates that enzyme leakage may be a marker for the assessment of freezability and fertility. Table 5 Modification of some enzymatic activity in buffalo semen in relation to freeze–thawing process Parameters Fresh semen Thawed semen Reference Ž . Hyaluronidase act. Urml 3.0 6.82 Akhtar et al., 1990 Ž . Asp. am. transf. act. Urml 214.06 1619.18 Bhosrekar et al., 1994 Ž . Lact. dehydr. act. Ur100 ml 5153.23 35,103.3 Bhosrekar et al., 1994 Ž . Alk. phosp. act. Ur100 ml 83.74 726.90 Bhosrekar et al., 1994 Ž . Acid phosp. act. Ur100 ml 58.21 521.36 Bhosrekar et al., 1994 Ž . GOTrGPT release mMrl 14.94 24.11 Dhami and Sahni, 1993 Ž . Ž . Oba et al. 1994 and Vale 1997 used the thermoresistance test to evaluate the Ž quality of thawed semen, examining the sperm motility at different times 0, 30, 60, 120, . 180 min during incubation at a temperature of 408C. When frozen–thawed semen is used for in vitro insemination, in the absence of uterine or tubal fluids that facilitate motility and enhances capacitation, the spermatozoa need to be capacitated in an artificial culture medium. Different commercial culture Ž . media were examined by Del Sorbo et al. 1992 . 5.6. Use of frozen–thawed semen for insemination and fertility results The fertility rate is considered to be the best parameter to assess the quality of Ž . frozen–thawed semen Vale, 1997 . However, a shortcoming of the assessment is that due to the high number of spermatozoa used for AI, variations in the state of sperm cells may not be reflected by the fertility rates. On the other hand, laboratory tests can indicate the extent of sperm damage during freeze–thawing, but they cannot accurately predict fertility of spermatozoa. The difficulty regarding AI in buffaloes is the application of the method in the field and not the semen technology. The most common problems are poor conditions of Ž . hygiene, detection of oestrus, method and time of insemination Vale, 1997 . Detection Ž of oestrus in buffaloes is problematic because of the scarce behavioural signs Ohashi, . 1994 and the muddy appearance of buffalo cows. These problems have been reported Ž . by authors from different countries Danell et al., 1984, Drost et al., 1985 . Zicarelli et Ž . al. 1997a have examined the role of teaser bulls in detection of estrus and fertility after AI. Cows run with a vasectomised bull had higher pregnancy rate than the control Ž . Ž females after AI in natural oestrus 42.5 vs. 18.9 and in induced oestrus of 51.1 . vs. 33.3 . According to the authors, buffaloes adapt with difficulty to an artificial breeding system which does not take into account their ethological needs. The low conception rates to AI in buffaloes could also be due to the small size of the uterine body in comparison to that of bovines. Because of this, the semen could inadvertently be introduced into one uterine horn and not into the body of uterus Ž . Ž . Zicarelli et al., 1997b . To clarify the situation, Zicarelli et al. 1997b compared cervical insemination with deposition of semen into the cranial section of the uterine horns, and obtained better fertility by the latter method when it was performed 24 h before ovulation. There was no difference between the two methods when the insemina- tions was performed after ovulation. Ž . Villa and Fabbri 1993 conducted two AI trials in farms located in three Italian w Ž . provinces using semen frozen in Laiciphos IMV extender and doses of insemination 6 Ž containing 8 = 10 live spermatozoa. The high variation in the conception rates 30.5– . 57.1 found for buffalo cows inseminated by the same method in all farms indicated a strong influence of environmental factors. Ž . Ž . Haranath et al. 1990 examined the efficiency of freezing in mini 0.25 ml and Ž . medium 0.5 ml straws using Murrah buffalo semen extended with a tris–yolk–glycerol Ž medium. The fertility rate was higher for packaging in mini than medium straws 52.7 . vs. 50.4 . The pregnancy rates in Surti buffaloes, after insemination with semen frozen in tris-, citrate- and lactose-based extenders were 42.7, 39.8 and 37.5, respectively Ž . Dhami and Kodagali, 1990 . Also in Surti buffaloes, semen frozen with a lactose– Ž . yolk–glycerol extender yielded 59.1 pregnancy rate Dhami et al., 1994 . Ž . In a further study Dhami and Sahni, 1994 comparing different cooling rates, the Ž . highest fertility rate 68.1 in Murrah buffaloes was obtained for semen cooled at a Ž . velocity of 0.28Crmin. El-Amrawi 1997 examined the fertility of semen thawed by Ž . different procedures and found that the best fertility rate 64.5 for semen thawed at Ž . 358C for 60 s. According to Vale 1997 , a pregnancy rate higher than 50 can be regarded as a good result after insemination with frozen–thawed buffalo semen.

6. Summary of procedure for freeze–thawing of semen