EFFECTS OF DIFFERENT TEMPERATURE TREATMENTS APPLIED TO DEEP STORED BULL SEMEN ON POST-THAW COLD SHOCKED SPERMATOZOA

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1 Bull Vet Inst Pulawy 50, 79-83, 2006 EFFECTS OF DIFFERENT TEMPERATURE TREATMENTS APPLIED TO DEEP STORED BULL SEMEN ON POST-THAW COLD SHOCKED SPERMATOZOA ZEKARIYA NUR, IRFAN KAMURAN ILERI 1 AND KEMAL AK 1 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Uludag, Gorukle, Bursa, Turkey 1 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Istanbul, Avcılar, Istanbul, Turkey nurzek@uludag.edu.tr Received for publication October 14, Abstract Various treatments were applied to frozen semen during storage in containers with different nitrogen level and the effects of temperature changes due to these treatments on the resistance of spermatozoa against post-thaw cold shock were studied. Straws with frozen bull semen were placed into two identical field containers. One of these containers was the low nitrogen level container with 3 cm nitrogen level that is 2 cm above the lower end of straws and the other one was the high nitrogen level container with 17 cm nitrogen level that is 2 cm above the upper end of straws. Canister No. 1 in both containers served as control without any manipulation. Canister No. 2 were raised up to the neck of the container and kept at this level for 10 s. Canister No. 3 were taken out to such a position that their base was at the entrance of the container and kept at this level for 20 s. These manipulations were repeated 100 times for canisters No. 2 and 3, in both containers. Motility, defected acrosome, and hypo-osmotic swelling test were carried out at post thaw (37ºC for 30 s) and after cold shock in a cold water bath (5-6ºC for 30 s). Postthaw cold-shock was observed to cause significant damage to motility, membrane integrity, and acrosome integrity of spermatozoa. Low nitrogen level was not capable of protecting viability and morphological structures of spermatozoa. In container with low nitrogen level, raising the canister from the container and keeping it at this level for 20 s made the spermatozoa more sensitive to cold shock. In conclusion, container nitrogen level and canister manipulations had negative effects on both shocked and unshocked spermatozoa. Also the results provide evidence that not only canisters misuse but also low nitrogen level made spermatozoa more sensitive to post-thaw cold shock. Key words: bulls, frozen semen, liquid nitrogen level, membrane integrity, acrosome integrity. The temperature fluctuation occurs during storage of frozen semen at 196 C as hazardous effects on sperm viability and morphology. Rising container temperature above 120 C leads to irreversible damage to spermatozoa (8). For thawing, the canisters with straws must be raised up to the neck of the tank and then lowered to the bottom. These manipulations cause temperature fluctuations in the straws remaining in the canisters. Because of the high surface-to-volume ratio, straws are significantly sensitive to temperature changes (8, 10, 15). Sequential thermal exposure during routine manipulations may lead to significant permanent damage to spermatozoa. The hazards of these sequential temperature fluctuations on straw frozen semen were lower in the container with full nitrogen level than in the container with low nitrogen level (8). Frozen semen is most frequently damaged during handling after thawing and prior to the insemination (8). Cold shock is the permanent injury to sperm caused by a sudden decrease in post-thaw semen temperature. There are many studies on the effects of post thaw cold shock on spermatozoon fertility and sperm morphology (1, 2, 4, 5, 11, 12), but there was not found any reported study on effects of temperature changes of semen frozen at 196 C on the resistance of spermatozoa against post-thaw cold shock. In this study, it was aimed to determine the effects of temperature changes on the motility, acrosome and membrane integrity; and resistance of spermatozoa against postthaw cold shock due to mishandling of frozen bull semen during storage. Material and Methods Animals and semen freezing. The semen from 4 Holstein bulls, 3-5 years of age, was used. Sperm was collected twice a week and semen with motility and concentration values higher than 70% and

2 80 600x10 6 spermatozoa/ml, respectively, was frozen in 0.25 ml French straws. Collected semen was extended with Laiciphos 488 at to obtain 25x10 6 spermatozoa/straw. Extended semen was cooled to 5 C in 45 min, and after glycerolization it was subjected to equilibration for 2 h. Semen was frozen at 10 C for 7 min and then stored in liquid nitrogen for several months. Container and nitrogen levels. Two identical field containers (Air Liquid GT 9) were used. One of them was the container with low (3 cm) liquid nitrogen level (LLN) and the other one was the container with high (17 cm) liquid nitrogen (HLN). Both nitrogen levels were 2 cm above upper ends of straws. Liquid nitrogen was added to both containers to maintain these levels every other day. Semen distribution and canister manipulations. Three canisters were used in each container. Between 39 and 57 straws from each bull were placed to each canister. Thus, approximately 200 straws were placed to each canister. A total of 1141straws were used. Canister No. 1 in both containers served as the control without any manipulation. Canister No. 2 were raised up to the neck of the container and kept at this level for 10 s. Canister No. 3 were taken out to such a position that its base was at the entrance of the container and kept at this level for 20 s. These manipulations were applied for 100 times to all canisters except controls. Thawing procedures. Once these canister manipulations were finished the canister was raised up to the neck of the container and each straw was taken out for 10 s. The straw frozen semen was randomly divided into two groups. In the first group (unshocked group) straw frozen semen was evaluated after thawing at 37 C for 30 s. In the second group (cold shocked group) straw frozen semen was thawed in a 37 C water bath for 30 s and then immediately plunged into an icedcold water bath at 5-7 C for 90 s to provide cold shock on spermatozoa. Then the cold shocked spermatozoa were placed into a 37 C water bath for 30 s to examine the sperm characteristics. Obtained semen parameters of each bull were separately recorded to evaluate the effects of bull factors on the parameters. Assessment of frozen-thawed semen. Postthaw sperm motility was assessed subjectively using a phase-contrast microscope (x400) with a warm slide (38 C). Acrosome integrity was evaluated using Hancock s formalin solution (6). One drop of thawed semen was diluted in 1 ml of 37 C Hancock s formalin solution and examined using a phase-contrast microscope at x1000 magnification under oil immersion. Thereafter, 200 spermatozoa were counted per a slide to determine the percentage of damaged acrosomes. Sperm membrane integrity was assessed by hypo-osmotic swelling (HOS) test as described by Jeyendran et al. (9) with some modifications. A volume of 20 µl of semen was added to 1 ml of the hypo-osmotic solution (100 mosm/l) composed of fructose and sodium citrate, and incubated at 37 C for 60 min. Immediately after the incubation, one drop of the semen was placed on a glass slide and covered with cover slide and evaluated under the phase-contrast microscope (x400). Microscope fields were selected randomly. At least 200 spermatozoa were counted per a slide and percentages of swollen tail spermatozoa were recorded. Statistical analysis. Calculated mean semen parameters were subjected to an arc sin transformation and then subjected to an analysis of variance (ANOVA, SPSS 10.0) using the General Linear Model (GLM). The statistical model included the effects of bull, canister manipulations, container nitrogen level and thawing procedure. Duncan test was used to examine the statistical significance of the differences, with P 0.05 considered to represent real differences. Results In terms of container nitrogen levels, canister manipulations, and thawing procedures, the results obtained from the analysis of the studied sperm parameters for spermatozoa thawed at 37 C for 30 s, and post-thaw cold shocked spermatozoa (at 5-7 C for 30s) are summarized in Table I. In both containers all the exmined semen parameters of unshocked spermatozoa were not generally affected by canister manipulations (P>0.05, Table 2). Also similar results were obtained in post-thaw cold shocked groups but only percentage of motility obtained in canister No. 3 were lower than canister No. 1 in the container with LLN (P<0.005, Table 1). It was observed that there were interactions among container nitrogen levels, thawing protocols, and canister manipulations on spermatozoon motility (P<0.05) and acrosome integrity (P<0.01, Table 2). Due to canister manipulations in both containers, defected acrosome rates of unshocked and shocked spermatozoa were higher in canisters No. 2 and 3 than in canister No. 1. Thawing protocols have an impact on the rates of motility (P<0.001), defected acrosome (P<0.001), and swollen tail spermatozoa (P<0.001) (Table 2). Regardless of container liquid nitrogen level, post-thaw cold shock application increased percentage of defected acrosome (Table 1). There was an interaction between thawing protocol and canister manipulations on acrosome integrity. Also it was found an interaction between thawing protocol and container nitrogen level on the percentages of motility and swollen tail spermatozoa (P<0.05, Table 2). In the container with HLN, percentage of motility and swollen tail spermatozoa were not affected by post-thaw cold shock application in general, but in the container with LLN the percentages of motility in canisters No. 2 and 3 and swollen tail spermatozoa in canisters No. 1 and 2 were affected by post-thaw cold application. The rates of motility, defected acrosome and swollen tail spermatozoa were affected by container

3 81 nitrogen level (P<0.001, Table 2). In terms of the same thawing protocol and canister manipulation, motility rates of spermatozoa in the container with HLN were higher than the motility rates of spermatozoa in the container with LLN (Table 1). Table 1 Mean sperm parameters in relation to liquid nitrogen level (LNL), canister position and thawing procedures LNL Low liquid nitrogen level High liquid nitrogen level No. of Thawing Defected n Motility canister procedures acrosome HOS test 1 37 C/ ±1.6 de 4.38±0.2 e 28.69±1.3 abc 2 37 C/ ±1.2 abcd 4.55±0.2 e 28.72±1.3 abc 3 37 C/ ±1.1 cde 4.74±0.2 e 27.52±1.5 bcd 1 cold shock ±1.3 ef 7.77±0.3 a 25.27±1.2 d 2 cold shock ±1.3 fg 7.08±0.3 ab 22.99±1.3 d 3 cold shock ±1.3 g 7.25±0.2 ab 24.50±1.3 cd 1 37 C/ ±1.6 ab 2.90±0.2 f 28.92±1.6 abc 2 37 C/ ±1.3 abc 2.91±0.2 f 31.46±1.3 ab 3 37 C/ ±1.1 a 3.82±0.2 e 33.40±1.5 a 1 cold shock ±1.6 abcd 5.91±0.3 cd 28.43±1.3 abc 2 cold shock ±1.2 bcd 6.79±0.3 bc 30.57±1.2 ab 3 cold shock ±1.3 abcd 5.78±0.2 d 30.35±1.3 ab abcdefg Values with different superscripts in the same colon are significantly different (P<0.05); ± SE. Table 2 Effect of thawing procedures (TP), liquid nitrogen level (LNL), canister position, bulls, and their influence on semen parameters (ANOVA) Factor Motility Defected acrosome Swollen tail spermatozoa TP *** *** *** LNL *** *** *** Canister NS NS NS Bull *** *** *** TP * LNL * NS * TP * canister NS *** NS TP *bull *** *** NS LNL * canister * NS NS LNL *bull *** NS NS Canister * bull NS NS * TP * canister * LNL * ** NS TP *bull * canister *** NS *** TP * LNL *bull NS * NS LNL * canister * bull *** NS *** TP * canister * LNL *bull ** NS NS * P<0.05, ** P<0.01, *** P<0.001, NS - not significant.

4 82 Motility rates of unshocked spermatozoa in canisters No. 1 and 2 in the container with HLN were higher than that of canister No. 1 in container with LLN (P<0.05). However, for cold shocked spermatozoa, there was a statistical difference between the same canister groups in both containers (P<0.05). Defected acrosome rates of unshocked and shocked spermatozoa were higher in all canisters of the container with LLN than of the container with HLN (P<0.05). Rates of swollen tail spermatozoa were lower in container with LLN than in container with HLN in all canisters for both thawing protocols (P<0.05) except canisters No. 1 and 2 of unshocked groups. Bull factors, affected all the investigated semen parameters (Table 2, P<0.001). Also, there were found some interactions among bulls, thawing procedures, and container nitrogen level on some semen parameters (Table 2). Discussion From collection to insemination, frozen semen is exposed to undesirable temperature changes during routine applications such as refilling the container liquid nitrogen, raising and lowering the canisters for artificial insemination, and both before thaw and post-thaw environment temperatures (8, 14). In this study, the effect of nitrogen level and temperature differences encountered by frozen bull semen during deep storage on sperm motility, acrosome and membrane integrity were investigated. It was observed that the examined semen parameters were not affected by canister manipulation in both containers, but there was found an interaction between thawing protocol and container nitrogen level on the percentages of motility and swollen tail spermatozoa (P<0.05, Table 2). The percentage of motility of cold shocked spermatozoa in canister No. 3 in the container with LLN was the lowest and significantly differed from canister No. 1 (P<0.05). In the container with LLN, raising the canister and keeping it at this level for 20 s made the spermatozoa more sensitive to cold shock. While percentage of motility and HOS in LLN were affected by cold shock application in some canisters, percentage of motility and HOS in HLN were not affected by cold shock application in all canisters. These results showed that effects of cold shock on frozen semen stored in LLN level were more severe due to canister manipulation. The canisters must be raised and lowered for insemination several times. Especially acrosome integrity, motility and morphology of frozen semen are affected by the frequency and duration of these manipulations (13). Also Uzey et al. (17) demonstrated that motility rate of liquid nitrogen stored semen was affected by canister mishandling. There were interactions among container nitrogen level, thawing protocol, and canister manipulations influencing the percentage of motility (P<0.05) and acrosome integrity (P<0.01, Table 2). Pace et al. (13) reported that rising and lowering straw frozen semen in liquid nitrogen container by 480 times reduced the motility and intact acrosome rates. They also reported that percentage of acrosomal defect of semen stored in the top goblet was higher than that of semen stored in the bottom goblet. It was observed that post-thaw semen parameters were not affected by canister manipulations; however, only percentage of motility of cold shocked spermatozoa in canister No. 3 in LLN container was affected by canister manipulations. The percentage of motility, defected acrosome, and swollen tail spermatozoa were affected by container nitrogen level (P<0.001, Table 2). Higher motility and swollen tail spermatozoa and lower defected acrosome rates were observed in container with HLN than in container with LLN for both thawing procedures. Senger (15) reported that keeping the frozen semen in the neck of the container for approximately 1 min increased temperature from 196 C to 180 C in the container full of liquid nitrogen. However, in low liquid nitrogen container (14 cm) temperature increased from 196 C to 124 C. Mishandling the frozen semen in field containers causes cumulative damages to frozen semen more severe than one accidentally event. Low liquid nitrogen has a negative effect on sperm characteristics. These negative effects are more harmful on post-thaw cold shocked spermatozoa. This study indicates that motility rates are affected by canister misuse and postthaw temperature changes in container with LLN. It is also reported that any increase in container temperature above 100 C leads to changes in ice crystal formation and re-crystallization occurs after transferring again the canister into liquid nitrogen of 196 C. Even these thermodynamic events occur at 130 C (14). Stanley et al. (16) reported that the glass transition temperature of ice occurred by warming the temperature above 130 C. This temperature warming results in rearrangement of ice formation that is known as re-crystallization. The recrystallization cause slight damage to the cryopreserved cells. Howard and Pace (8) reported that any increase in container temperature above 120 C leads to irreversible damage to frozen spermatozoa. In the present study, regardless of the container nitrogen level, post-thaw cold shock application increased the percentage of defected acrosome. Percentage of motility and swollen tail spermatozoa were not affected by the cold shock in the container with HLN. Thawing protocols were found to have an important impact on the rates of motility (P<0.001), defected acrosome (P<0.001), and swollen tail spermatozoa (P<0.001, Table 3). In both containers the motility and swollen tail spermatozoa of cold shocked groups were lower than those of unshocked groups. It was also observed that in both containers cold shock application increased rates of defected acrosome (P<0.05). The common point of view among many scientists is that the exposure of thawed spermatozoa to different temperatures impairs their morphological characteristics, plasma membrane integrity (7), and fertilizing capability of spermatozoa (5). Post-thaw

5 83 fertilizing ability of spermatozoa is greatly affected by protocols such as thawing temperature and duration (1, 10). In many studies it was reported that thawing at 37 C for 30 s was the best thawing procedure for frozen bull spermatozoa (3, 4, 11, 12, 15). It was also reported that experimental post-thaw cold shock impaired the survival of frozen-thawed semen (2, 7, 11, 12). In the present study swollen tail spermatozoa rates were affected by nitrogen level and cold shock applications (P<0.01). However, canister manipulations did not affect rates of swollen tail spermatozoa. It was observed that the HOS test was inconvenient to determine the effect of temperature changes on spermatozoa during deep temperature storage. This is in agreement with the result reported by Holt and North (7) who found that plasma membrane integrity was maintained throughout the cooling and freezing process even in the absence of cryoprotectants and the membrane damage occurred during post-thaw rewarming within 2 C and 30 C temperature ranges. The results reported here provide evidence that post-thaw cold shock could cause significant damage to acrosomal integrity in all canisters stored in both containers. Regardless of container nitrogen level, canister manipulations increased the percentage of defected acrosome. LLN had a hazardous effect on the percentages of motility of both shocked and unshocked groups except canister No. 2 of unshocked group. Also the percentage of swollen tail spermatozoa of LLN container was found lower than HLN container except of canisters No. 1 and 2 of unshocked groups. While the percentage of motility of unshocked groups in canisters No. 1 and 3 of container with LLN were similar, the percentages of motility of post-thaw cold shocked were different. Thus, it could be concluded that sequential temperature fluctuations in container with LLN did not affect the post-thaw motility but made spermatozoa more sensitive to post-thaw cold shock. Container nitrogen level and canister manipulations had negative effects on both shocked and unshocked spermatozoa. Also the results reported here provided evidence that not only canisters misuse but also low nitrogen level made spermatozoa more sensitive to post-thaw cold shock. References 1. Bell M., Wang R., Hellstrom W.J.G., Sikka S.C.: Effect of cryoprotective additives and cryopreservation protocol on sperm membrane lipid peroxidation and recovery of motile human sperm. J Androl 1993, 14, Brown J.L., Senger P.L., Hillers J.K.: Influence of thawing time and post-thawing time and post-thaw temperature on acrosomal maintenance and motility of bovine spermatozoa in 0.5 ml French straws. J Anim Sci 1982, 54, Correa J.A., Heersch G., Zavos P.M.: Sperm membrane functional integrity and response of frozen-thawed bovine spermatozoa during the swelling test incubation at varying temperatures. Theriogenology 1997, 47, Correa J.R., Rodriguez M.C., Patterson D.J., Zavos P.M.: Thawing and processing of cryopreserved bovine spermatozoa at various temperatures and their effects on sperm viability, osmotic shock and sperm membrane functional integrity. Theriogenology 1996, 46, De Leeuw F.E., Chen H-C., Colenbrander B., Verkleij A.J.: Cold-induced ultra structural changes in bull and boar sperm plasma membranes. Cryobiology 1990, 27, Hancock J.L.: The morphology of bull spermatozoa. J Exp Biol 1952, 29, Holt W.V., North R.D.: Effects of temperature and restoration of osmotic equilibrium during thawing on the induction of plasma membrane damage in cryopreseved ram spermatozoa. Biol Reprod 1994, 51, Horward T.H., Pace M.M.: Seminal evaluation and artificial insemination In: Fertility and Infertility in Veterinary Practice. Edited by Laing J.A., Bailliere Tindal, London, 1988, pp Jeyendran R.S., Van der Ven H.H., Perez-Pelaez M., Crabo B.G., Zaneveld L.J.D.: Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fert 1984, 70, Johnson M.S., Senger P.L., Hancock D.D., Alexander B.M., Sasser R.G.: Fertility of bull semen packaged in 0.25ml and 0.5 ml French straws. J. Anim. Sci 1995, 73, Nur Z., Dogan I., Soylu M.K., Ak K.: Effect of different thawing procedures on quality of bull semen. Rev Med Vet 2003, 154, Nur Z., Sagirkaya H., Dogan I., Soylu M.K., Ak K., Ileri İ.K.: Effect of low temperature thawing procedure and post-thaw cold shock on frozen bull semen. Medycyna Wet 2005, 61, Pace M.M., Sullivan J.J., Elliott F.I., Graham E.F., Coulter G.H.: Effects of thawing temperature, number of spermatozoa and spermatozoal quality on fertility of bovine spermatozoa packaged in 5 ml French straws. J Anim Sci 1981, 53, Pickett B.W., Berndtson W.E.: Procedures for handling frozen bovine semen in the field. In: Current Therapy in Theriogenology: Diagnosis, Treatment and Prevention of Reproductive Diseases in Animals. Edited by Morrow D.A., W.B. Saunders Company, Philadelphia London Toronto, 1980, pp Senger P.L.: Handling frozen bovine semen-factors which influence viability and fertility. Theriogenology 1980, 13, Stanley P.L., Helen M.P., Roger G.G.: Cryopreservation of human spermatozoa, In: Current Practices and Controversies in Assisted Reproduction. Edited by Vayena E., Rove P.J, Griffin P.D., World Health Organization, Geneva, 2001, pp Uzey N., Ak K. Ileri I.K.: Konteynerde saklanan boga spermasina cevre faktorlerinin etkileri. I U Vet Fak Derg 2000, 26,