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Morphological Changes in Chicken and Turkey Spermatozoa Incubated Under Various Conditions1,2
Morphological Changes in Chicken and Turkey Spermatozoa Incubated Under Various Conditions1'2 R. N. CLARKE Department of Poultry Science, University of Maryland, College Park, Maryland 20742 M. R. BAKST USDA, SEA-AR, Avian Physiology Laboratory, Beltsville, Maryland 20705 M. A. OTTINGER
(Received for publication August 16, 1982) ABSTRACT The viability and morphology of undiluted and diluted chicken and turkey spermatozoa were compared when incubated at either 4 1 , 25, 15, or 5 C for 0 (control), 3, or 6 hr. Increasing the incubation time to 6 hr, raising the incubation temperature to 41 C, or both resulted in higher numbers of dead spermatozoa in semen from both species. Conversely, dilution (1:5 with Beltsville Poultry Semen Extender) of chicken or turkey semen resulted in a significant (P<.05) decrease in the number of dead spermatozoa as compared to their respective undiluted counterparts, especially in samples incubated at 41 C. Turkey semen exhibited a somewhat lower percentage of dead spermatozoa, bent spermatozoa, and sperm tail coiling than did chicken semen incubated under similar conditions. No significant (P>.05) differences were observed in chicken and turkey semen samples when measuring other sperm cell abnormalities regardless of incubation conditions. These results indicate selective species differences in sperm morphological changes in response to incubation temperature, incubation time, and semen dilution. (Key words: chicken, turkey, spermatozoa, morphology, light microscopy, undiluted semen, diluted semen) 1984 Poultry Science 63:801-805 INTRODUCTION
Chicken and turkey spermatozoa show similar patterns of cellular disruption when held in both the frozen and liquid states (Bakst and Sexton, 1979). These patterns include plasmalemma distention, which precedes other common sperm anomalies such as bent spermatozoa (also referred to as neck-bent or crooked-neck spermatozoa), tail coiling, and midpiece disruption (Wakely and Kosin, 1951; Bakst and Sexton, 1979). Such damage has been attributed to adverse osmotic conditions and has been observed in spermatozoa from both the chicken (Yamane, 1972; Van Wambeke, 1977; Bakst and Sexton, 1979; Bakst, 1980) and the turkey (Marquez and Ogasawara, 1977; Bakst, 1980). Nevertheless, diluted
'Scientific Article No. A-3266, Contribution No. 6338 of the Maryland Agriculture Experiment Station (Department of Poultry Science). 2 Submitted in part for fulfillment of requirements of Master of Science degree, University of Maryland.
chicken semen has been preserved at 2 to 5 C for up to 24 hr with no loss in fertilizing capacity (Van Wambeke, 1970, 1972; Sexton, 1978). Similar storage procedures with turkey spermatozoa have resulted in a severe loss in fertilizing capacity during short-term (1 to 2 hr), low-temperature (5 to 15 C) storage (Graham and Brown, 1971; Litjen, 1972). Furthermore, aeration of semen samples has allowed the storage of chicken and turkey spermatozoa for 48 and 24 hr, respectively, with a resultant hen fertility of greater than 90% of that of freshly-diluted semen (Wishart, 1981). Because artificial insemination is extensively used in the turkey industry, it is important to characterize the basis of these variable responses to incubation procedures. In this study, a comparison has been made of the percentage dead and morphology of undiluted and diluted chicken and turkey spermatozoa subjected to varying incubation temperatures and holding times. Such observations may contribute to our understanding of the underlying mechanisms of these varied
801
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 10, 2014
Department of Poultry Science, University of Maryland, College Park, Maryland 20742
CLARKE ET AL.
802
responses observed in spermatozoa subjected to short-term storage in vitro. MATERIALS AND METHODS
RESULTS AND DISCUSSION
Turkey semen exhibited a somewhat lower percentage of dead spermatozoa than did chicken semen incubated under similar conditions. Additionally, changes in incubation temperature, time, and the addition of diluent had significant (P<.05) effects on the percentage of dead spermatozoa in both chicken (Fig. la) and turkey (Fig lb) semen samples. Lengthening the time of incubation resulted in increased levels of dead spermatozoa at all incubation temperatures, although this effect
CHICKEN SEMEN Undiluted
2 40
3 20
41 5 15 25 INCUBATION TEMPERATURE (C)
TURKEY SEMEN Undiluted
15
25
41
5
15
25
41
INCUBATION TEMPERATURE (C)
FIG. 1. The effect of semen dilution, incubation time, and incubation temperature on the percentage of dead spermatozoa in chicken and turkey semen.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 10, 2014
Shaver broiler breeder males between 3 3 and 48 weeks of age were caged individually and maintained on a 14L:10D hour photoperiod. They had free access to water and a standard breeder diet. Nicholas Large White turkey males between 45 and 50 weeks of age were kept in floor pens, maintained on a 12L:12D hour photoperiod, and also had free access to water and a standard breeder diet. Semen samples were collected by abdominal massage (Burrows and Quinn, 1937) from males chosen at random from the respective flocks and pooled by species. One-tenth milliliter aliquots of undiluted or .5 ml aliquots of chicken or turkey semen diluted 1 part semen to 5 parts Beltsville Poultry Semen Extender (BPSE), osmolarity 355 (Sexton, 1978), were placed in 15-ml conical test tubes and incubated in a water bath at either 41, 25, 15, or 5 C. Examination of semen incubated under various treatment conditions for 3 or 6 hr was conducted by taking aliquots from the same storage vessel; semen were gently mixed prior to each sampling period. All samples were evaluated for percentage of dead spermatozoa and for abnormalities in sperm morphology. The determination of the number of dead spermatozoa was done by using a nigrosin-eosin preparation (Ernst and Ogasawara, 1970). The comparative protocols for chicken and turkey semen were somewhat different. One drop of chicken semen was gently mixed with 12 drops of the nigrosin-eosin stain and allowed to incubate for 1 min at room temperature. For turkey semen, 1 drop of semen was mixed with 20 drops of stain and incubated for 2 minutes. One large drop of either mixture was placed on the far end of an oil-free microscopic slide and a smear was prepared (Ernst and Ogasawara, 1970). The resulting preparation was dried quickly and completely with warm air and examined under oil immersion bright field conditions. A total of 200 sperm cells were examined at random, and those showing any red or pink discoloration were assumed dead at the time the stain was introduced. An additional 200 sperm cells (alive or dead) were examined and characterized as normal or as having one or more of the following morphological abnormalities: absent or bulbous
acrosome, nuclear swelling, knotted or bent spermatozoa, disrupted or swollen mitochondria, and coiled or lost tail. Six replicates were performed for both chicken and turkey spermatozoa. In all cases, the absolute numbers of dead or abnormal spermatozoa were converted to a percentage. An analysis of variance was performed on all data with sources of variation corresponding to a factorial (4 temperature levels, 2 dilution levels, 2 incubation times). Orthogonal polynomial contrasts were also computed to describe linear and quadratic effects, as well as their interactions. All statistical tests were performed at a = .05.
803
MORPHOLOGY OF CHICKEN AND TURKEY SPERMATOZOA
Most of the sperm abnormalities characterized during this study, including sperm tail coiling and bent spermatozoa, have previously been found to be primarily due to osmotic damage (Yamane, 1972; Marquez and Ogasawara, 1977; Van Wambeke, 1977; Bakst and Sexton, 1979; Bakst, 1980). In previous studies, the osmolarities of undiluted chicken and turkey semen were 323 and 339 mOsm, respectively (Graham and Brown, 1971), while the osmolarity of the BPSE added to semen in this study was 355 mOsm. However, extenders with osmolarities ranging between 300 to 450 mOsm were found to have no detrimental effects on the fertilizing capacity of chicken and turkey spermatozoa (Harris et ah, 1963; Hobbs and Harris, 1963a,b; Graham and Brown, 1971; Bakst, 1980). Similarly, small changes in the osmolarity of the surrounding medium also appeared to have little effect on
the percentage of sperm abnormalities. That is, the use of extender was not associated with increased numbers of abnormal spermatozoa. Bent spermatozoa is a frequendy encountered abnormality of chicken and turkey semen that has been attributed to semen dilution, lowering of semen temperature, and adverse osmotic conditions. Saeki (1960) and Tsukunaga (1971) observed an increase in the number of bent spermatozoa upon lowering semen temperature, while Saeki (1960) found an increase in the percentage of bent spermatozoa after semen dilution. In this study, dilution of chicken and turkey semen with BPSE (which is isotonic to semen) did not result in a significant increase in bent spermatozoa (Fig. 2). In contrast, increasing the incubation temperature of chicken spermatozoa resulted in elevated numbers of bent spermatozoa (Fig. 2a). No such effect was observed in turkey semen samples. Lengthening the time of incubation from 3 to 6 hr resulted in substantially higher levels of bent spermatozoa in both chicken and turkey semen. As seen in the measurement of other sperm anomalies, chicken and turkey semen incubated for 6 hr had significantly (P<.05) higher levels of sperm tail coiling than in samples held for 3
CHICKEN SEMEN Undiluted
.
Diluted
< 2
5
15
25 41 5 15 25 INCUBATION TEMPERATURE (C)
TURKEY SEMEN Undiluted
Diluted
10
15
25 41 5 15 25 INCUBATION TEMPERATURE (C)
FIG. 2. The effect of semen dilution, incubation time, and incubation temperature on the percentage of bent spermatozoa in chicken and turkey semen.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 10, 2014
was most pronounced in samples held at 41 C. There were significant (P<.05) linear and quadratic effects associated with incubation temperatures in both species. The percentage of dead spermatozoa remained relatively constant in samples held at or below 25 C, while raising the incubation temperature above this level (up to 41 C) resulted in substantially more dead spermatozoa. This effect was more pronounced in undiluted semen samples, where there were higher numbers of dead spermatozoa when compared to their undiluted counterparts. The elevated number of dead spermatozoa in undiluted chicken and turkey semen held at 41 C may have been due to a higher respiratory rate of spermatozoa held at this temperature (Clarke et ah, 1982). Consequently, undiluted semen incubated at 41 C over a 6-hr period could have been expected to undergo substrate depletion, a change in pH, and an increase in the concentration of metabolic by-products, which directly or indirectly could lead to sperm death. These detrimental conditions were at least partially alleviated by the addition of the extender. A relatively small rise in the percentage of dead spermatozoa was found when increasing incubation temperature of diluted semen samples as compared to undiluted samples. The analysis of viability data from both species revealed significant (P<.05) time X temperature and dilution X temperature interactions. The relative increase in the percentage of dead spermatozoa in samples incubated at 41 C for 6 hr was greater than in those held for 3 hr.
CLARKE ET AL.
804 CHICKEN SEMEN Undiluted
Diluted 6hr
p 203hr
6hr
£ 10
3hr
25
41
5
15
25
INCUBATION TEMPERATURE (C) TURKEY SEMEN Undiluted
Diluted
25
41
5
15
25
41
INCUBATION TEMPERATURE (C)
FIG. 3. The effect of semen dilution, incubation time, and incubation temperature on the percentage of sperm tail coiling in chicken and turkey semen.
hr (Fig. 3). The effect of lengthening incubation time was more pronounced in semen held at higher temperatures. Both linear and quadratic effects were associated with incubation temperature. A significant (P<.05) increase in the percentage of sperm tail coiling was measured in samples held at 41 C as compared to those incubated at lower temperatures. This effect was more prominent in undiluted chicken semen (Fig. 3a), whereas no dilution effect was seen in turkey semen samples (Fig. 3b). Furthermore, significant dilution X temperature and time X temperature interactions were found in the analysis of data from both chicken and turkey semen samples. Although no significant differences were found when measuring other sperm abnormalities, the percentages of bent spermatozoa and tail coiling were generally higher in chicken semen than in turkey semen incubated under identical conditions. These results are similar to those found when measuring sperm viability. In a separate study in which semen from both species was incubated under the identical conditions imposed during the present study, diluted chicken and turkey sperm fertilizing capacities were highest at 5 C when held for 6 hr (75 and 24%, respectively; Clarke et al,
ACKNOWLEDGMENTS The authors wish to express appreciation to D. M. Gavalek and G. Kaminski for their technical assistance and to L. Douglass and E. Russek for their advice on statistical analyses. REFERENCES Bakst, M. R., 1980. Fertilizing capacity and morphology of fowl and turkey spermatozoa in hypotonic extender. J. Reprod. Fertil. 60: 121-127. Bakst, M. R„ and T. J. Sexton, 1979. Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing. J. Reprod. Fertil. 5 5 : 1 - 7 . Burrows, W. J., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16:19-24. Clarke, R. N., T. J. Sexton, and M. A. Ottinger, 1982.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 10, 2014
R 20-
1982). If data from the two studies are assumed comparable, then it appears that the levels of dead spermatozoa, bent spermatozoa, and sperm tail coiling did not influence the fertilizing capacity of chicken and turkey spermatozoa when incubated at 5 C for 6 hr. This is in agreement with data presented by Sampson and Warren (1939) who concluded that morphologic defects of fowl spermatozoa were not serious causes of infertility. In contrast, a relationship between bent spermatozoa and fertilizing capacity was proposed by Saeki (1960) and appeared to be dependent on the number of bent spermatozoa in the semen sample. Current data, as well as findings from other studies (Van Wambeke, 1977; Clarke et al, 1982), suggest that fertility (of hens) was not affected when bent spermatozoa comprised less than 15% of the sperm population. However, a decrease in hen fertility was observed when bent spermatozoa comprised 37% of the sperm population (Saeki, 1960). In a separate study, hens were inseminated (200 million sperm per artificial insemination dose; Clarke et al, 1982) with semen held under the same conditions employed in the present set of experiments. Based on data from the present experiment, sperm morphology was not a reliable indicator of the fertilizing capacity of either chicken or turkey spermatozoa. However, overall abnormal spermatozoa in the incubated semen samples was generally less than 20%. Presumably, the effect of an abnormal sperm population on fertilizing capacity would increase proportionally with a decrease in the number of spermatozoa used per insemination.
MORPHOLOGY OF CHICKEN AND TURKEY SPERMATOZOA
Sampson, I. R., and D. C. Warren, 1939. Density of suspension and morphology of sperm in relation to fertility in fowl. Poultry Sci. 18:301-307. Sexton, T. J., 1978. A new poultry semen extender. I. Effect of extension on the fertility of chicken semen. Poultry Sci. 56:1443 — 1446. Tsukunaga, S., 1971. Morphological evidence of temperature shock in fowl sperm (preliminary report). Bull. Hiroshima Agric. Coll. 4:145-153. Van Wambeke, F., 1970. The effect of two insemination methods on the fertilizing capacity of fowl semen stored for 24 and 48 hours. Pages 349-351 in Proc. 14th World Poult. Congr. Van Wambeke, F., 1972. Fertility and hatchability results with fowl spermatozoa in fresh and freeze-dried diluent. Br. Poult. Sci. 13:179-183. Van Wambeke, F., 1977. The effect of toxicity of storage media for fowl semen on the occurrence of neck-bending spermatozoa, fertility, and hatchability. Br. Poult. Sci. 18:163-168. Wakely, W. J., and I. L. Kosin, 1951. A study of the morphology of the spermatozoa — with special reference to the seasonal prevalence of abnormal types. Am. J. Vet. Res. 12:240-245. Winer, B. J., 1971. Statistical Principles in Experimental Design. 2nd ed. McGraw-Hill Book Co., New York, NY. Wishart, G. J., 1981. The effect of continuous aeration on the fertility of fowl and turkey semen stored above OC. Br. Poult. Sci. 22:445-450. Yamane, J., 1972. A peculiar deformation of cock sperm, neck-bending, caused by osmo- and thermo-shock, and its significance to artificial insemination of fowl. Pages 1681—1684 in Proc. 7th Int. Congr. Anim. Reprod. Artif. Insem., Munich.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 10, 2014
Effects of holding temperature and storage time on respiratory rate, motility, and fertility of chicken and turkey semen. Poultry Sci. 6 1 : 1912-1918. Ernst, R. A., and F. X. Ogasawara, 1970. Alive-dead stain to test poultry semen quality. One Sheet Answers, Univ. Calif., Agric. Ext. Serv. Graham, E. F., and K. I. Brown, 1971. Effect of osmotic pressure of semen extenders on the fertility and hatchability of turkey eggs. Poultry Sci. 50:836-838. Harris, G. C , Jr., T. D. Hobbs, J. E. Brown, and L. B. Warren, 1963. The storage of turkey spermatozoa in sodium citrate and C 0 2 extenders. Poultry Sci. 42:536-538. Hobbs, T. D„ and G. C. Harris, 1963a. Effect of freezing point depression and pH on motility and fertility of chicken spermatozoa stored in sodium citrate extenders. Poultry Sci. 42:254—259. Hobbs, T. D., and G. C. Harris, 1963b. Effect of freezing point depression and C 0 2 on motility and fertility of chicken spermatozoa stored in carbon dioxide extenders. Poultry Sci. 42: 388-393. Litjen, J. B., 1972. Artificial insemination in turkeys and an investigation into a suitable diluent for turkey semen. Pages 16—18 in Proc. 8th Int. Congr. Anim. Reprod. Artif. Insem., Krakow. Marquez, B., and F. Ogasawara, 1977. Ultrastructural changes in turkey spermatozoa after immersion in glycerolyzed media and during various steps used in cryopreservation. Poultry Sci. 56:1806— 1813. Saeki, Y., 1960. Crooked-necked spermatozoa in relation to low fertility in the artifical insemination of fowl. Poultry Sci. 39:1354-1361.
805
(Received for publication August 16, 1982) ABSTRACT The viability and morphology of undiluted and diluted chicken and turkey spermatozoa were compared when incubated at either 4 1 , 25, 15, or 5 C for 0 (control), 3, or 6 hr. Increasing the incubation time to 6 hr, raising the incubation temperature to 41 C, or both resulted in higher numbers of dead spermatozoa in semen from both species. Conversely, dilution (1:5 with Beltsville Poultry Semen Extender) of chicken or turkey semen resulted in a significant (P<.05) decrease in the number of dead spermatozoa as compared to their respective undiluted counterparts, especially in samples incubated at 41 C. Turkey semen exhibited a somewhat lower percentage of dead spermatozoa, bent spermatozoa, and sperm tail coiling than did chicken semen incubated under similar conditions. No significant (P>.05) differences were observed in chicken and turkey semen samples when measuring other sperm cell abnormalities regardless of incubation conditions. These results indicate selective species differences in sperm morphological changes in response to incubation temperature, incubation time, and semen dilution. (Key words: chicken, turkey, spermatozoa, morphology, light microscopy, undiluted semen, diluted semen) 1984 Poultry Science 63:801-805 INTRODUCTION
Chicken and turkey spermatozoa show similar patterns of cellular disruption when held in both the frozen and liquid states (Bakst and Sexton, 1979). These patterns include plasmalemma distention, which precedes other common sperm anomalies such as bent spermatozoa (also referred to as neck-bent or crooked-neck spermatozoa), tail coiling, and midpiece disruption (Wakely and Kosin, 1951; Bakst and Sexton, 1979). Such damage has been attributed to adverse osmotic conditions and has been observed in spermatozoa from both the chicken (Yamane, 1972; Van Wambeke, 1977; Bakst and Sexton, 1979; Bakst, 1980) and the turkey (Marquez and Ogasawara, 1977; Bakst, 1980). Nevertheless, diluted
'Scientific Article No. A-3266, Contribution No. 6338 of the Maryland Agriculture Experiment Station (Department of Poultry Science). 2 Submitted in part for fulfillment of requirements of Master of Science degree, University of Maryland.
chicken semen has been preserved at 2 to 5 C for up to 24 hr with no loss in fertilizing capacity (Van Wambeke, 1970, 1972; Sexton, 1978). Similar storage procedures with turkey spermatozoa have resulted in a severe loss in fertilizing capacity during short-term (1 to 2 hr), low-temperature (5 to 15 C) storage (Graham and Brown, 1971; Litjen, 1972). Furthermore, aeration of semen samples has allowed the storage of chicken and turkey spermatozoa for 48 and 24 hr, respectively, with a resultant hen fertility of greater than 90% of that of freshly-diluted semen (Wishart, 1981). Because artificial insemination is extensively used in the turkey industry, it is important to characterize the basis of these variable responses to incubation procedures. In this study, a comparison has been made of the percentage dead and morphology of undiluted and diluted chicken and turkey spermatozoa subjected to varying incubation temperatures and holding times. Such observations may contribute to our understanding of the underlying mechanisms of these varied
801
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 10, 2014
Department of Poultry Science, University of Maryland, College Park, Maryland 20742
CLARKE ET AL.
802
responses observed in spermatozoa subjected to short-term storage in vitro. MATERIALS AND METHODS
RESULTS AND DISCUSSION
Turkey semen exhibited a somewhat lower percentage of dead spermatozoa than did chicken semen incubated under similar conditions. Additionally, changes in incubation temperature, time, and the addition of diluent had significant (P<.05) effects on the percentage of dead spermatozoa in both chicken (Fig. la) and turkey (Fig lb) semen samples. Lengthening the time of incubation resulted in increased levels of dead spermatozoa at all incubation temperatures, although this effect
CHICKEN SEMEN Undiluted
2 40
3 20
41 5 15 25 INCUBATION TEMPERATURE (C)
TURKEY SEMEN Undiluted
15
25
41
5
15
25
41
INCUBATION TEMPERATURE (C)
FIG. 1. The effect of semen dilution, incubation time, and incubation temperature on the percentage of dead spermatozoa in chicken and turkey semen.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 10, 2014
Shaver broiler breeder males between 3 3 and 48 weeks of age were caged individually and maintained on a 14L:10D hour photoperiod. They had free access to water and a standard breeder diet. Nicholas Large White turkey males between 45 and 50 weeks of age were kept in floor pens, maintained on a 12L:12D hour photoperiod, and also had free access to water and a standard breeder diet. Semen samples were collected by abdominal massage (Burrows and Quinn, 1937) from males chosen at random from the respective flocks and pooled by species. One-tenth milliliter aliquots of undiluted or .5 ml aliquots of chicken or turkey semen diluted 1 part semen to 5 parts Beltsville Poultry Semen Extender (BPSE), osmolarity 355 (Sexton, 1978), were placed in 15-ml conical test tubes and incubated in a water bath at either 41, 25, 15, or 5 C. Examination of semen incubated under various treatment conditions for 3 or 6 hr was conducted by taking aliquots from the same storage vessel; semen were gently mixed prior to each sampling period. All samples were evaluated for percentage of dead spermatozoa and for abnormalities in sperm morphology. The determination of the number of dead spermatozoa was done by using a nigrosin-eosin preparation (Ernst and Ogasawara, 1970). The comparative protocols for chicken and turkey semen were somewhat different. One drop of chicken semen was gently mixed with 12 drops of the nigrosin-eosin stain and allowed to incubate for 1 min at room temperature. For turkey semen, 1 drop of semen was mixed with 20 drops of stain and incubated for 2 minutes. One large drop of either mixture was placed on the far end of an oil-free microscopic slide and a smear was prepared (Ernst and Ogasawara, 1970). The resulting preparation was dried quickly and completely with warm air and examined under oil immersion bright field conditions. A total of 200 sperm cells were examined at random, and those showing any red or pink discoloration were assumed dead at the time the stain was introduced. An additional 200 sperm cells (alive or dead) were examined and characterized as normal or as having one or more of the following morphological abnormalities: absent or bulbous
acrosome, nuclear swelling, knotted or bent spermatozoa, disrupted or swollen mitochondria, and coiled or lost tail. Six replicates were performed for both chicken and turkey spermatozoa. In all cases, the absolute numbers of dead or abnormal spermatozoa were converted to a percentage. An analysis of variance was performed on all data with sources of variation corresponding to a factorial (4 temperature levels, 2 dilution levels, 2 incubation times). Orthogonal polynomial contrasts were also computed to describe linear and quadratic effects, as well as their interactions. All statistical tests were performed at a = .05.
803
MORPHOLOGY OF CHICKEN AND TURKEY SPERMATOZOA
Most of the sperm abnormalities characterized during this study, including sperm tail coiling and bent spermatozoa, have previously been found to be primarily due to osmotic damage (Yamane, 1972; Marquez and Ogasawara, 1977; Van Wambeke, 1977; Bakst and Sexton, 1979; Bakst, 1980). In previous studies, the osmolarities of undiluted chicken and turkey semen were 323 and 339 mOsm, respectively (Graham and Brown, 1971), while the osmolarity of the BPSE added to semen in this study was 355 mOsm. However, extenders with osmolarities ranging between 300 to 450 mOsm were found to have no detrimental effects on the fertilizing capacity of chicken and turkey spermatozoa (Harris et ah, 1963; Hobbs and Harris, 1963a,b; Graham and Brown, 1971; Bakst, 1980). Similarly, small changes in the osmolarity of the surrounding medium also appeared to have little effect on
the percentage of sperm abnormalities. That is, the use of extender was not associated with increased numbers of abnormal spermatozoa. Bent spermatozoa is a frequendy encountered abnormality of chicken and turkey semen that has been attributed to semen dilution, lowering of semen temperature, and adverse osmotic conditions. Saeki (1960) and Tsukunaga (1971) observed an increase in the number of bent spermatozoa upon lowering semen temperature, while Saeki (1960) found an increase in the percentage of bent spermatozoa after semen dilution. In this study, dilution of chicken and turkey semen with BPSE (which is isotonic to semen) did not result in a significant increase in bent spermatozoa (Fig. 2). In contrast, increasing the incubation temperature of chicken spermatozoa resulted in elevated numbers of bent spermatozoa (Fig. 2a). No such effect was observed in turkey semen samples. Lengthening the time of incubation from 3 to 6 hr resulted in substantially higher levels of bent spermatozoa in both chicken and turkey semen. As seen in the measurement of other sperm anomalies, chicken and turkey semen incubated for 6 hr had significantly (P<.05) higher levels of sperm tail coiling than in samples held for 3
CHICKEN SEMEN Undiluted
.
Diluted
< 2
5
15
25 41 5 15 25 INCUBATION TEMPERATURE (C)
TURKEY SEMEN Undiluted
Diluted
10
15
25 41 5 15 25 INCUBATION TEMPERATURE (C)
FIG. 2. The effect of semen dilution, incubation time, and incubation temperature on the percentage of bent spermatozoa in chicken and turkey semen.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 10, 2014
was most pronounced in samples held at 41 C. There were significant (P<.05) linear and quadratic effects associated with incubation temperatures in both species. The percentage of dead spermatozoa remained relatively constant in samples held at or below 25 C, while raising the incubation temperature above this level (up to 41 C) resulted in substantially more dead spermatozoa. This effect was more pronounced in undiluted semen samples, where there were higher numbers of dead spermatozoa when compared to their undiluted counterparts. The elevated number of dead spermatozoa in undiluted chicken and turkey semen held at 41 C may have been due to a higher respiratory rate of spermatozoa held at this temperature (Clarke et ah, 1982). Consequently, undiluted semen incubated at 41 C over a 6-hr period could have been expected to undergo substrate depletion, a change in pH, and an increase in the concentration of metabolic by-products, which directly or indirectly could lead to sperm death. These detrimental conditions were at least partially alleviated by the addition of the extender. A relatively small rise in the percentage of dead spermatozoa was found when increasing incubation temperature of diluted semen samples as compared to undiluted samples. The analysis of viability data from both species revealed significant (P<.05) time X temperature and dilution X temperature interactions. The relative increase in the percentage of dead spermatozoa in samples incubated at 41 C for 6 hr was greater than in those held for 3 hr.
CLARKE ET AL.
804 CHICKEN SEMEN Undiluted
Diluted 6hr
p 203hr
6hr
£ 10
3hr
25
41
5
15
25
INCUBATION TEMPERATURE (C) TURKEY SEMEN Undiluted
Diluted
25
41
5
15
25
41
INCUBATION TEMPERATURE (C)
FIG. 3. The effect of semen dilution, incubation time, and incubation temperature on the percentage of sperm tail coiling in chicken and turkey semen.
hr (Fig. 3). The effect of lengthening incubation time was more pronounced in semen held at higher temperatures. Both linear and quadratic effects were associated with incubation temperature. A significant (P<.05) increase in the percentage of sperm tail coiling was measured in samples held at 41 C as compared to those incubated at lower temperatures. This effect was more prominent in undiluted chicken semen (Fig. 3a), whereas no dilution effect was seen in turkey semen samples (Fig. 3b). Furthermore, significant dilution X temperature and time X temperature interactions were found in the analysis of data from both chicken and turkey semen samples. Although no significant differences were found when measuring other sperm abnormalities, the percentages of bent spermatozoa and tail coiling were generally higher in chicken semen than in turkey semen incubated under identical conditions. These results are similar to those found when measuring sperm viability. In a separate study in which semen from both species was incubated under the identical conditions imposed during the present study, diluted chicken and turkey sperm fertilizing capacities were highest at 5 C when held for 6 hr (75 and 24%, respectively; Clarke et al,
ACKNOWLEDGMENTS The authors wish to express appreciation to D. M. Gavalek and G. Kaminski for their technical assistance and to L. Douglass and E. Russek for their advice on statistical analyses. REFERENCES Bakst, M. R., 1980. Fertilizing capacity and morphology of fowl and turkey spermatozoa in hypotonic extender. J. Reprod. Fertil. 60: 121-127. Bakst, M. R„ and T. J. Sexton, 1979. Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing. J. Reprod. Fertil. 5 5 : 1 - 7 . Burrows, W. J., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16:19-24. Clarke, R. N., T. J. Sexton, and M. A. Ottinger, 1982.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 10, 2014
R 20-
1982). If data from the two studies are assumed comparable, then it appears that the levels of dead spermatozoa, bent spermatozoa, and sperm tail coiling did not influence the fertilizing capacity of chicken and turkey spermatozoa when incubated at 5 C for 6 hr. This is in agreement with data presented by Sampson and Warren (1939) who concluded that morphologic defects of fowl spermatozoa were not serious causes of infertility. In contrast, a relationship between bent spermatozoa and fertilizing capacity was proposed by Saeki (1960) and appeared to be dependent on the number of bent spermatozoa in the semen sample. Current data, as well as findings from other studies (Van Wambeke, 1977; Clarke et al, 1982), suggest that fertility (of hens) was not affected when bent spermatozoa comprised less than 15% of the sperm population. However, a decrease in hen fertility was observed when bent spermatozoa comprised 37% of the sperm population (Saeki, 1960). In a separate study, hens were inseminated (200 million sperm per artificial insemination dose; Clarke et al, 1982) with semen held under the same conditions employed in the present set of experiments. Based on data from the present experiment, sperm morphology was not a reliable indicator of the fertilizing capacity of either chicken or turkey spermatozoa. However, overall abnormal spermatozoa in the incubated semen samples was generally less than 20%. Presumably, the effect of an abnormal sperm population on fertilizing capacity would increase proportionally with a decrease in the number of spermatozoa used per insemination.
MORPHOLOGY OF CHICKEN AND TURKEY SPERMATOZOA
Sampson, I. R., and D. C. Warren, 1939. Density of suspension and morphology of sperm in relation to fertility in fowl. Poultry Sci. 18:301-307. Sexton, T. J., 1978. A new poultry semen extender. I. Effect of extension on the fertility of chicken semen. Poultry Sci. 56:1443 — 1446. Tsukunaga, S., 1971. Morphological evidence of temperature shock in fowl sperm (preliminary report). Bull. Hiroshima Agric. Coll. 4:145-153. Van Wambeke, F., 1970. The effect of two insemination methods on the fertilizing capacity of fowl semen stored for 24 and 48 hours. Pages 349-351 in Proc. 14th World Poult. Congr. Van Wambeke, F., 1972. Fertility and hatchability results with fowl spermatozoa in fresh and freeze-dried diluent. Br. Poult. Sci. 13:179-183. Van Wambeke, F., 1977. The effect of toxicity of storage media for fowl semen on the occurrence of neck-bending spermatozoa, fertility, and hatchability. Br. Poult. Sci. 18:163-168. Wakely, W. J., and I. L. Kosin, 1951. A study of the morphology of the spermatozoa — with special reference to the seasonal prevalence of abnormal types. Am. J. Vet. Res. 12:240-245. Winer, B. J., 1971. Statistical Principles in Experimental Design. 2nd ed. McGraw-Hill Book Co., New York, NY. Wishart, G. J., 1981. The effect of continuous aeration on the fertility of fowl and turkey semen stored above OC. Br. Poult. Sci. 22:445-450. Yamane, J., 1972. A peculiar deformation of cock sperm, neck-bending, caused by osmo- and thermo-shock, and its significance to artificial insemination of fowl. Pages 1681—1684 in Proc. 7th Int. Congr. Anim. Reprod. Artif. Insem., Munich.
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Effects of holding temperature and storage time on respiratory rate, motility, and fertility of chicken and turkey semen. Poultry Sci. 6 1 : 1912-1918. Ernst, R. A., and F. X. Ogasawara, 1970. Alive-dead stain to test poultry semen quality. One Sheet Answers, Univ. Calif., Agric. Ext. Serv. Graham, E. F., and K. I. Brown, 1971. Effect of osmotic pressure of semen extenders on the fertility and hatchability of turkey eggs. Poultry Sci. 50:836-838. Harris, G. C , Jr., T. D. Hobbs, J. E. Brown, and L. B. Warren, 1963. The storage of turkey spermatozoa in sodium citrate and C 0 2 extenders. Poultry Sci. 42:536-538. Hobbs, T. D„ and G. C. Harris, 1963a. Effect of freezing point depression and pH on motility and fertility of chicken spermatozoa stored in sodium citrate extenders. Poultry Sci. 42:254—259. Hobbs, T. D., and G. C. Harris, 1963b. Effect of freezing point depression and C 0 2 on motility and fertility of chicken spermatozoa stored in carbon dioxide extenders. Poultry Sci. 42: 388-393. Litjen, J. B., 1972. Artificial insemination in turkeys and an investigation into a suitable diluent for turkey semen. Pages 16—18 in Proc. 8th Int. Congr. Anim. Reprod. Artif. Insem., Krakow. Marquez, B., and F. Ogasawara, 1977. Ultrastructural changes in turkey spermatozoa after immersion in glycerolyzed media and during various steps used in cryopreservation. Poultry Sci. 56:1806— 1813. Saeki, Y., 1960. Crooked-necked spermatozoa in relation to low fertility in the artifical insemination of fowl. Poultry Sci. 39:1354-1361.
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