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Effects of Holding Temperature and Storage Time on Respiratory Rate, Motility, and Fertility of Chicken and Turkey Semen1
Effects of Holding Temperature and Storage Time on Respiratory Rate, Motility, and Fertility of Chicken and Turkey Semen1 R. N. CLARKE, 2 T. J. SEXTON,3 and M. A. OTTINGER 2 Department of Poultry Science, University of Maryland, College Park, Maryland 20742, and Avian Physiology Laboratory, ARS, USDA, Beltsville, Maryland 20705 (Received for publication November 30, 1981)
1982 Poultry Science 61:1912-1917 INTRODUCTION
oxygen u p t a k e in chicken sperm was f o u n d t o be five times higher when i n c u b a t e d at 4 1 C t h a n at 5 C (Ashizawa and Nishiyama, 1 9 7 8 ) , whereas oxygen u p t a k e in t u r k e y sperm was severely diminished a t 5 C b u t n o t significantly different when incubated at 15, 2 5 , or 35 C (Cherms, 1968). T h e objective of this study was t o characterize respiration, m o t i l i t y , and fertility of chicken and t u r k e y sperm held for 0, 3, or 6 hr at 4 1 , 2 5 , 1 5 , o r 5 C.
Researchers have successfully preserved diluted chicken semen at low t e m p e r a t u r e s (2 t o 5 C) for up t o 2 4 h r (Van W a m b e k e , 1970, 1 9 7 2 ; S e x t o n , 1 9 7 8 ) . However, this has n o t been possible with t u r k e y semen; several workers reported loss in fertilizing capacity during short-term (1 to 2 h r ) l o w t e m p e r a t u r e (5 t o 15 C) storage ( G r a h a m and Brown, 1 9 7 1 ; Litjen, 1972). With t h e e x c e p t i o n of studies on frozen semen (Bakst and Sexton, 1979) few studies have c o m p a r e d t h e physiology and biochemistry of chicken and t u r k e y semen treated identically. Data indicate t h a t t h e metabolism of washed t u r k e y sperm differs significantly from washed chicken sperm (Lake, 1 9 7 1 ; S e x t o n , 1974). F u r t h e r m o r e , Sexton ( 1 9 7 4 ) found t h a t t u r k e y sperm were m o r e efficient metabolically t h a n chicken sperm because of their lower e n d o g e n o u s respiratory rate, higher oxidation rate of e x o g e n o u s substrates, and lower accumulation of lactic acid u n d e r aerobiosis. In separate studies, t h e rate of
MATERIALS AND METHODS
1 Scientific Article No. A3069, Contribution No. 6134, of Maryland Experiment Station (Department of Poultry Science). 2 Department of Poultry Science. 3 Avian Physiology Laboratory.
Individually caged male broiler breeders (Shaver strain), b e t w e e n 33 and 4 8 weeks of age, and dwarf broiler breeder h e n s (Shaver strain), b e t w e e n 4 3 and 50 weeks of age, were maintained o n a 1 4 L : 1 0 D p h o t o p e r i o d . Both males and females were fed a standard 16% protein breeder diet o n a restricted basis of 93 and 115 g / b i r d / d a y , respectively. Male t u r k e y s of t h e Nicholas Large White variety, between 45 and 6 0 weeks of age, were maintained on a 12L: 12D p h o t o p e r i o d . Females (Nicholas Large White and D i a m o n d H y b r i d Small White), 3 4 t o 4 1 weeks of age, were maintained on a 14L: 10D p h o t o p e r i o d . Both males and females were given feed and water ad libitum.
1912
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ABSTRACT A series of experiments was conducted to measure the respiratory activity, motility, and fertility of chicken and turkey sperm in undiluted and diluted (1 part semen to 5 parts Beltsville Poultry Semen Extender) semen held at either 41, 25, 15, or 5 C for 3 or 6 hr. Sperm respiration was temperature dependent, increasing with increasing temperature. When incubated under identical conditons, the respiratory rate of spermatozoa in diluted semen of both species was significantly (P<.05) higher than in undiluted semen. In undiluted and diluted chicken and turkey semen, sperm motility was lowest at 41 C. No differences in the motility of spermatozoa from undiluted and diluted semen of either species were observed in the unstored controls. Sperm motility in samples held at 15 or 5 C was similar to that of the unstored controls. The fertility of diluted chicken semen was highest (75%) after 6 hr of storage at 5 C. Fertility levels (71 to 83%) of chicken semen held for 3 hr at 25, 15, or 5 C did not differ from the unstored control. In contrast, the fertilizing capacity of diluted turkey semen (0 to 45%) was significantly lower than the unstored controls (68%), regardless of holding times and temperatures. The fertility of undiluted turkey semen was significantly (P<.05) lower than the unstored control in all cases, with the exception of semen held at 25 C for 3 hr. (Key words: semen, chicken, turkey, storage)
HOLDING CHICKEN AND TURKEY SEMEN
tility. Eight milliliters of semen were collected weekly from each species, and sperm concentrations were determined with a KlettSummerson Photoelectric Colorimeter (Cecil, 1981). The weekly insemination dose (volume) for each sample was adjusted to contain 200 million sperm. Six hens were assigned per treatment group. Diamond Hybrid Small White and Nicholas Large White turkey hens were inseminated with diluted and undiluted semen, respectively. Inseminations were performed weekly for seven consecutive weeks, and eggs were candled on day 7 of incubation to determine fertility. Early dead embryos were identified by candling, but clear eggs were not broken-out for further examination. Fertile eggs were transferred to hatching machines, and any unhatched chicken eggs at 21 days and turkey eggs at 28 days were considered "late dead" embryos. All experiments were arranged as a 2 X 2 X 4 X 2 factorial, the factors being species, dilution, temperature, and time, respectively. After applying a square root transformation, respiration data were analyzed by a split plot-repeated measure design, with species, dilution, and temperature as the main effects and time as the subplot (Winer, 1971). Completely random designs were used for both the sperm motility and fertility experiments. Motility data were analyzed by a four-way analysis of variance (ANOVA) and fertility by a
TABLE 1. Cumulative oxygen consumption of chicken and turkey sperm incubated at various temperatures and times
Semen Chicken Undiluted Diluted Turkey Undiluted Diluted
Incubation time (hr)
Ml of 0 2 uptake/10 8 sperm 1 41 C
25 C
15 C
5C
3 6 3 6
15.04 a 'S 20.19 a > f 17.49 a 'g 37.44a>e
7 89b,c,h 9.52 b .g 13.69 b . f 20.99 b . e
.47c-g .95c>g 2.15c>t 5.49c>e
-6.29d.f -7.17d-f -4.36d.e -4.50 d > e
3 6 3 6
7.56 a . h * 11.47 a .g* 32.81a>f 54.02 a . e
3.75 b . h 6.67 b .g 11.21 b . f 21.37 b . e
1.29c.g 3.27c.g 4.26 c . f 9.17 c . e
-4.71d.f -5.19 d > f -4.19d.f -2.99 d > e -2.54 d > e
' ' ' Means with different superscripts within rows differ significantly (P<.05). ' * ' Means with different superscripts within columns differ significantly (P<.05). 1
All values are means of 15 determinations (pooled SEM = ± 1.107) except for those indicated by asterisks, which are means of 13 determinations.
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Semen of both species was collected by abdominal massage (Burrows and Quinn, 1937). For each experiment, semen samples were pooled from at least 5 birds chosen at random from their respective flocks. Within each of the experiments, two independent trials were conducted with respect to species. Experiment 1-. Determination of Sperm Respiration. Fifty microliters of 20% KOH were added to the center well of each incubation vessel to absorb metabolic C 0 2 . Pooled semen was divided into .1 ml (undiluted) and .5 ml aliquots. The latter was diluted (1:5) with Beltsville Poultry Semen Extender (BPSE) (Sexton, 1978). Flasks containing semen were placed on one of two Gilson Differential Respirometers, Model GP-14 for the 41 and 25 C incubations and Model GR-14 for the 15 and 5 C incubations. Oxygen uptake readings were taken at hourly intervals for 6 hr. Experiment 2: Evaluation of Sperm Motility. Progressive motility of sperm cells (movement in a forward motion) was estimated on a percentage basis by the method of Sexton (1976) on undiluted and diluted samples within 15 min of semen collection (controls) and after being held at 4 1 , 25, 15, or 5 C for either 3 or 6 hr. One drop of semen was placed on a microscope slide (maintained at room temperature) and examined immediately by phase contrast microscopy (40 x ) . Experiment 3: Measurement of Egg Fer-
1913
CLARKE ET AL.
1914
B
60
^ J
TURKEY 41 C
50
40 J CHICKEN 41 C 30 CHICKEN 41 C
r
r-'^
—T
T-"~r 2
3
4
1 CHICKEN 25 C J TURKEY 25 C
j TURKEY 25 C " * " ] CHICKEN 25 C
> -<
20 TURKEY 41 C 10
5 INCUBATION TIME (hr :
(hr )
.
B
J TURKEY 15 C [ CHICKEN 15 C
•
""-"---v - J
...-J_-
-4
1 - — "T— 2
3
4
INCUBATION TIME (hr )
i—
—-r-
_. j
TURKEY 5 C
— J CHICKEN 5 C
3 4 5 INCUBATION TIME (hr )
PIG. 1. The respiratory rate of undiluted (A) and diluted (B) chicken and turkey sperm incubated at various temperatures. (Mean and SEM of 6 determinations.)
weighted ANOVA, data being weighted by the square root of the number of eggs laid per hen. Differences between treatment means were determined using the Student-Newman-Keuls multiple comparison test in all cases. Pooled standard errors of the mean (SEM) were calculated from the ANOVA. RESULTS
Experiment 1. The respiration of chicken and turkey spermatozoa decreased significantly (P<.05) as the incubation temperature was lowered from 41 to 5 C for all treatments. No oxygen was taken up by sperm when incubated for 3 or 6 hr at 5 C (Table 1). With the exception of diluted chicken semen incubated at 41 C for 3 hr, respiration of chicken and turkey sperm in diluted semen was significantly (P<.05) higher than in undiluted semen incubated under similar conditions. The cumulative oxygen consumption by spermatozoa in undiluted chicken or turkey semen ceased
after 3 hr of incubation at 15 C but increased significantly between 3 and 6 hr of incubation at 41 o r 2 5 C . With undiluted semen, chicken sperm respired at a rate higher than undiluted turkey sperm when incubated at 41 C under similar conditions, but there was no difference at 25 or 15 C (Fig. la). With diluted semen, turkey sperm respired at a higher rate than chicken sperm after 1 hr of incubation at 41 C, but no differences were observed at 25 or 15 C (Fig. lb). Experiment 2. The percentage of progressively motile chicken and turkey sperm held at 41 C was significantly (P<.05) lower than the value for controls and for those held at 25, 15, or 5 C (Table 2). With the exception of undiluted chicken semen held for 6 hr at 15 C and 5 C, sperm motility of samples held at these temperatures did not differ significantly (P>.05) from that of the unstored controls. At 25 C, diluted chicken semen held for 3 hr was the
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INCUBATION TIME
HOLDING CHICKEN AND TURKEY SEMEN
1915
TABLE 2. Percent ofprogessively motile chicken and turkey spermatozoa held at various temperatures and incubation times
Inculbation time (hr)
Semen Chicken Undiluted Diluted Turkey Undiluted
3 6 3 6
Control
41 C
25 C
84.3 a . e
12.1 c . e . f 7 d,f 25.0b,e 17.8c>e 9.3 c > e . f .7Cf 14.3 c . e 5.0C,e,f
87.1 a . e
93.6 a . e 93.6 a ' e
ISC
5C
57.8 b . f .g 42.2 c .g 77.1a.e 65.7 b . e .g
77.8 a . e 65.0b,e 82.8 a . e 77.8 a . e
80.0 a . e 70.0 b ,e 87.9 a . e 83.6 a > e
80.0 b . e 74.3 b . e 60.7 b > f 41.4 b .g
89.3 a . b > e 88.6 a . e 92.1 a - e 90.0 a . e
92.1 a - e 87.1 a > e 92.1a.e 89.3 a > e
' ' ' Means with different superscripts within rows plus control differ significantly (P<.05). ans with different superscripts within columns differ significantly (P<.05). 1
All values are means of 15 determinations (pooled SEM = ± 5.556).
only sample t h a t did n o t differ from t h e controls. Sperm motility of neither species was affected b y semen dilution when i n c u b a t e d at 15 and 5 C. A higher level of chicken sperm motility was n o t e d w h e n diluted and stored at 25 C, whereas t u r k e y sperm held at this temperature was n o t affected by semen dilution. Sperm motility of t h e c o n t r o l semen samples from b o t h species was unaffected b y dilution. There was a significant ( P < . 0 5 ) species-dilution
interaction with respect t o sperm m o t i l i t y . Experiment 3. T h e fertility of u n d i l u t e d chicken semen held for 3 (41%) and 6 h r (28%) at 5 C was higher t h a n t h a t of samples held at higher t e m p e r a t u r e s b u t significantly l o w e r t h a n t h e u n s t o r e d control ( 8 5 % ; T a b l e 3). Regardless of die holding t e m p e r a t u r e , t h e fertilizing capacity of u n d i l u t e d chicken semen was severely diminished after 3 and 6 hr. T h e fertility of diluted chicken semen held for 3 h r at 2 5 , 15, and 5 C was n o t significantly dif-
TABLE 3. Percent weighted fertility of chicken and turkey sperm held at various temperatures and incubation times
Incubation time (hr)
Semen Chicken Undiluted Diluted Turkey Undiluted Diluted
Percent weighted fertility 1 Control
3 6 3 6
85.0 a . e
3 6 3 6
80.0 a . e
81.0 a . e
68.0a>f
41 C
Od.f 0 d,f 2ld,e 3d,f ld,e ld,e 0 d ,e 0 d ,e
25 C
15C
10 d ,c,g l d ,g 79a'e 61c-f
18 c .g 6 d,h 83 a - e 6 4 b,c,f
4 1 b,f c
82 a > c 56 b ,f 45 b .g 12d,h
68 b > e 55b,f 20 c .g ld,h
21c.f 39 c ' e 29 d ,c,f 24C.f
a,b,c,d Means with different superscripts within rows plus control differ significantly (P<.05). e,f,g,h 1
Means with different superscripts within columns differ significantly (P<.05).
All values are means of 7 weekly determinations (pooled SEM = + 4.497).
5C
28 >g 71a-e 75a.b.e
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Diluted
3 6 3 6
Percent progressive motility 1
CLARKE ET AL.
1916
DISCUSSION
That semen dilution increased chicken and turkey sperm respiration was not surprising and was probably due to an increased availability of nutrients, such as fructose, or was increased by dilution per se, The BPSE contains 5 mg/ml of fructose, a level 100 times greater than that found in undiluted semen (Kamar and Rizik, 1972). Thus, the added fructose from BPSE could account for the higher respiratory activity. Furthermore, semen dilution has been shown to stimulate sperm metabolism (Sexton, 1976), and this phenomenon has been related to an increase in enzyme activity associated with the tricarboxylic acid (TCA) cycle (Smith etal, 1957). In diluted semen, the respiratory rate of turkey sperm was higher than that of chicken sperm at 41 C, which is in contradiction to earlier reports comparing the respiration of washed chicken and turkey spermatozoa. Sexton (1974) found washed turkey sperm to have lower endogenous and exogenous (as determined by the addition of sugar and amino acids) respiratory rates than chicken sperm when incubated at 41 C. One possible explanation for the conflicting results is that the respiratory rate of turkey sperm may be
negatively influenced by the technique of washing. That turkey sperm are more fragile than chicken sperm is quite evident from the data in Table 3. Three independent trials were performed in an attempt to establish the cause of the negative respiratory readings at 5 C incubation. In the first trial, addition of antibacterials (see Sexton et ah, 1980) to the incubation medium did not have a significant effect on respiration, which indicates that oxygenproducing bacteria were not a factor. The influence of chemical breakdown products formed from extender solutes and the water solvent was studied in Trial 2. Heat treatment was used to accelerate chemical decomposition. Chemical degradations were not responsible for the negative readings. In Trial 3, a comparison of respirometers was made at 25 C by incubating vessels containing only BPSE. Both respirometers performed identically under these conditions, ruling out the possibility of respirometer malfunction. Therefore, the question of what caused the negative respiration readings at 5 C remains unanswered. It has generally been thought that a reduction in sperm metabolism is required to hold semen in vitro successfully without seriously decreasing its fertilizing ability. This concept appears to be true for chicken semen but not for turkey semen. Both undiluted and diluted turkey semen held at 15 or 5 C exhibited low fertility even though sperm respiratory activity was minimized. Perhaps factors other than low metabolic activity are needed for successful preservation of turkey semen. Semen dilution increased chicken sperm motility, whereas dilution per^ se had little influence on the level of motility of turkey sperm. This indicated that there were rather distinct differences in die effects of BPSE or dilution on the motility of chicken and turkey sperm held at low temperatures. It appears that the motilities of chicken and turkey sperm were stimulated when semen was diluted with BPSE and held at higher temperatures (41 and 25 C). This trend was also evident with chicken sperm held at the lower temperatures (15 and 5 C). However, semen dilution did not affect the motility of turkey sperm at 15 or 5 C. The low fertilizing capacity of diluted turkey sperm, both in the unstored control and treated groups, could have been related to either the extender, holding conditions, or
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ferent from that of the unstored control. There was no decrease in the fertility of diluted chicken semen held at 5 C for 6 hr when compared to the control. At all other holding temperatures, fertility levels were lower than those of the control. The fertilizing capacities of the unstored diluted (81%) and undiluted (85%) controls for chicken semen did not differ. Conversely, die fertilizing capacity of the diluted control turkey semen was significantly lower (68%) than that of the undiluted control (80%). The fertility of diluted turkey semen held at 25 and 15 C for 3 or 6 hr was significantly lower than the fertility of undiluted samples held under similar conditions. Chicken and turkey sperm held at 41 C showed consistently lower fertility levels than sperm held at the lower temperatures. The fertility of diluted and undiluted turkey semen held at 5 C for 3 or 6 hr was lower than that of the unstored controls. There were significant (P<.05) interactions of species-dilution, species-temperature, and temperature-holding time when the fertility data were analyzed.
HOLDING CHICKEN AND TURKEY SEMEN b o t h . It m a y be t h a t one or m o r e c o n s t i t u e n t s of t h e e x t e n d e r failed t o provide a suitable e n v i r o n m e n t to maintain o p t i m u m fertility levels for t u r k e y sperm. T h e dilution effect, which was characterized by an increase in respiratory rate of t u r k e y sperm (Table 1), could have c o n t r i b u t e d t o t h e decrease in fertility (Table 3) as has been reported with m a m m a l i a n sperm (for review see Sexton, 1979). T h u s , it remains uncertain as t o w h e t h e r BPSE or t h e dilution rate of 1:5 caused t h e decline in t h e fertility of t u r k e y semen.
T h e a u t h o r s wish t o t h a n k D. M. Gavelek for her technical assistance and L. Douglass for his statistical advice.
REFERENCES Ashizawa, K., and H. Nishiyama, 1978. Effects of temperature on the vigour of motility, oxygen consumption, and duration of motility of fowl spermatozoa under aerobic conditions. Japanese Poultry Sci. 14:264-266. Bakst, M. R., and T. J. Sexton, 1979. Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing. J. Reprod. Fert. 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. Cecil, H. C , 1981. Effects of dietary protein on body weight and reproductive performance of male turkeys. Poultry Sci. 1049-1055. Cherms, F. L., 1968. Variations in semen quality and the relationship of semen quality to fertility in turkeys. Poultry Sci. 47:746-754. 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. Kamar, G. A., and M. A. Risik, 1972. Semen characteristics of two breeds of turkeys. J. Reprod. Fertil. 29:317-325. Lake, P. E., 1971. Physiology and biochemistry of poultry semen. Pages 93—128 in Advances in Reproductive Physiology. M.W.H. Bishop, ed. Academic Press, New York, NY. Litjen, J. B., 1972. Artificial insemination in turkey and an investigation into a suitable diluent for turkey semen. 8th Int. Congr. Anim. Reprod. Artif. Insem. 3:1698-1701. Sexton, T. J., 1974. Oxidative and glycolytic activity of chicken and turkey spermatozoa. Comp. Biochem. Physiol. 48B:59-65. Sexton, T. J., 1976. Studies on the dilution of turkey semen. Br. Poultry Sci. 17:179-186. Sexton, T. J., 1978. A new poultry semen extender. 3. Effect of storage conditions on the fertilizing capacity of chicken semen stored at 5 C. Poultry Sci. 47:285-289. Sexton, T. J., 1979. Preservation of poultry semen. A review. Pages 159—170 in Beltsville Symposium in Agricultural Research. 3. Animal Reproduction. Allanheld, Osmun & Co., Montclair, NJ. Sexton, T. J., L. A. Jacobs, and G. R. McDaniel, 1980. A new poultry semen extender. 4. Effects of antibacterials in control of bacterial contamination in chicken semen. Poultry Sci. 59: 274-281. Smith, J. T., D. T. Mayer, and C. P. Merilan, 1957. Seasonal variation in the succinic dehydrogenase activity of bovine spermatozoa. J. Dairy Sci. 40:516-520. Van Wambeke, F., 1970. The effect of two insemination methods on the fertilizing capacity of fowl semen stored for 24 and 48 hrs. Pages 349—351 in Proc. 14th World's Poultry Congr. Van Wambeke, F., 1972. Fertility and hatchability results with fowl spermatozoa stored in fresh and freeze-dried diluent. Br. Poultry Sci. 13:179— 183. Winer, B. J., 1971. Statistical Principles in Experimental Design. 2nd ed. McGraw-Hill Book Co., New York, NY.
Downloaded from http://ps.oxfordjournals.org/ at University of Otago on December 22, 2014
ACKNOWLEDGMENTS
1917
1982 Poultry Science 61:1912-1917 INTRODUCTION
oxygen u p t a k e in chicken sperm was f o u n d t o be five times higher when i n c u b a t e d at 4 1 C t h a n at 5 C (Ashizawa and Nishiyama, 1 9 7 8 ) , whereas oxygen u p t a k e in t u r k e y sperm was severely diminished a t 5 C b u t n o t significantly different when incubated at 15, 2 5 , or 35 C (Cherms, 1968). T h e objective of this study was t o characterize respiration, m o t i l i t y , and fertility of chicken and t u r k e y sperm held for 0, 3, or 6 hr at 4 1 , 2 5 , 1 5 , o r 5 C.
Researchers have successfully preserved diluted chicken semen at low t e m p e r a t u r e s (2 t o 5 C) for up t o 2 4 h r (Van W a m b e k e , 1970, 1 9 7 2 ; S e x t o n , 1 9 7 8 ) . However, this has n o t been possible with t u r k e y semen; several workers reported loss in fertilizing capacity during short-term (1 to 2 h r ) l o w t e m p e r a t u r e (5 t o 15 C) storage ( G r a h a m and Brown, 1 9 7 1 ; Litjen, 1972). With t h e e x c e p t i o n of studies on frozen semen (Bakst and Sexton, 1979) few studies have c o m p a r e d t h e physiology and biochemistry of chicken and t u r k e y semen treated identically. Data indicate t h a t t h e metabolism of washed t u r k e y sperm differs significantly from washed chicken sperm (Lake, 1 9 7 1 ; S e x t o n , 1974). F u r t h e r m o r e , Sexton ( 1 9 7 4 ) found t h a t t u r k e y sperm were m o r e efficient metabolically t h a n chicken sperm because of their lower e n d o g e n o u s respiratory rate, higher oxidation rate of e x o g e n o u s substrates, and lower accumulation of lactic acid u n d e r aerobiosis. In separate studies, t h e rate of
MATERIALS AND METHODS
1 Scientific Article No. A3069, Contribution No. 6134, of Maryland Experiment Station (Department of Poultry Science). 2 Department of Poultry Science. 3 Avian Physiology Laboratory.
Individually caged male broiler breeders (Shaver strain), b e t w e e n 33 and 4 8 weeks of age, and dwarf broiler breeder h e n s (Shaver strain), b e t w e e n 4 3 and 50 weeks of age, were maintained o n a 1 4 L : 1 0 D p h o t o p e r i o d . Both males and females were fed a standard 16% protein breeder diet o n a restricted basis of 93 and 115 g / b i r d / d a y , respectively. Male t u r k e y s of t h e Nicholas Large White variety, between 45 and 6 0 weeks of age, were maintained on a 12L: 12D p h o t o p e r i o d . Females (Nicholas Large White and D i a m o n d H y b r i d Small White), 3 4 t o 4 1 weeks of age, were maintained on a 14L: 10D p h o t o p e r i o d . Both males and females were given feed and water ad libitum.
1912
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ABSTRACT A series of experiments was conducted to measure the respiratory activity, motility, and fertility of chicken and turkey sperm in undiluted and diluted (1 part semen to 5 parts Beltsville Poultry Semen Extender) semen held at either 41, 25, 15, or 5 C for 3 or 6 hr. Sperm respiration was temperature dependent, increasing with increasing temperature. When incubated under identical conditons, the respiratory rate of spermatozoa in diluted semen of both species was significantly (P<.05) higher than in undiluted semen. In undiluted and diluted chicken and turkey semen, sperm motility was lowest at 41 C. No differences in the motility of spermatozoa from undiluted and diluted semen of either species were observed in the unstored controls. Sperm motility in samples held at 15 or 5 C was similar to that of the unstored controls. The fertility of diluted chicken semen was highest (75%) after 6 hr of storage at 5 C. Fertility levels (71 to 83%) of chicken semen held for 3 hr at 25, 15, or 5 C did not differ from the unstored control. In contrast, the fertilizing capacity of diluted turkey semen (0 to 45%) was significantly lower than the unstored controls (68%), regardless of holding times and temperatures. The fertility of undiluted turkey semen was significantly (P<.05) lower than the unstored control in all cases, with the exception of semen held at 25 C for 3 hr. (Key words: semen, chicken, turkey, storage)
HOLDING CHICKEN AND TURKEY SEMEN
tility. Eight milliliters of semen were collected weekly from each species, and sperm concentrations were determined with a KlettSummerson Photoelectric Colorimeter (Cecil, 1981). The weekly insemination dose (volume) for each sample was adjusted to contain 200 million sperm. Six hens were assigned per treatment group. Diamond Hybrid Small White and Nicholas Large White turkey hens were inseminated with diluted and undiluted semen, respectively. Inseminations were performed weekly for seven consecutive weeks, and eggs were candled on day 7 of incubation to determine fertility. Early dead embryos were identified by candling, but clear eggs were not broken-out for further examination. Fertile eggs were transferred to hatching machines, and any unhatched chicken eggs at 21 days and turkey eggs at 28 days were considered "late dead" embryos. All experiments were arranged as a 2 X 2 X 4 X 2 factorial, the factors being species, dilution, temperature, and time, respectively. After applying a square root transformation, respiration data were analyzed by a split plot-repeated measure design, with species, dilution, and temperature as the main effects and time as the subplot (Winer, 1971). Completely random designs were used for both the sperm motility and fertility experiments. Motility data were analyzed by a four-way analysis of variance (ANOVA) and fertility by a
TABLE 1. Cumulative oxygen consumption of chicken and turkey sperm incubated at various temperatures and times
Semen Chicken Undiluted Diluted Turkey Undiluted Diluted
Incubation time (hr)
Ml of 0 2 uptake/10 8 sperm 1 41 C
25 C
15 C
5C
3 6 3 6
15.04 a 'S 20.19 a > f 17.49 a 'g 37.44a>e
7 89b,c,h 9.52 b .g 13.69 b . f 20.99 b . e
.47c-g .95c>g 2.15c>t 5.49c>e
-6.29d.f -7.17d-f -4.36d.e -4.50 d > e
3 6 3 6
7.56 a . h * 11.47 a .g* 32.81a>f 54.02 a . e
3.75 b . h 6.67 b .g 11.21 b . f 21.37 b . e
1.29c.g 3.27c.g 4.26 c . f 9.17 c . e
-4.71d.f -5.19 d > f -4.19d.f -2.99 d > e -2.54 d > e
' ' ' Means with different superscripts within rows differ significantly (P<.05). ' * ' Means with different superscripts within columns differ significantly (P<.05). 1
All values are means of 15 determinations (pooled SEM = ± 1.107) except for those indicated by asterisks, which are means of 13 determinations.
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Semen of both species was collected by abdominal massage (Burrows and Quinn, 1937). For each experiment, semen samples were pooled from at least 5 birds chosen at random from their respective flocks. Within each of the experiments, two independent trials were conducted with respect to species. Experiment 1-. Determination of Sperm Respiration. Fifty microliters of 20% KOH were added to the center well of each incubation vessel to absorb metabolic C 0 2 . Pooled semen was divided into .1 ml (undiluted) and .5 ml aliquots. The latter was diluted (1:5) with Beltsville Poultry Semen Extender (BPSE) (Sexton, 1978). Flasks containing semen were placed on one of two Gilson Differential Respirometers, Model GP-14 for the 41 and 25 C incubations and Model GR-14 for the 15 and 5 C incubations. Oxygen uptake readings were taken at hourly intervals for 6 hr. Experiment 2: Evaluation of Sperm Motility. Progressive motility of sperm cells (movement in a forward motion) was estimated on a percentage basis by the method of Sexton (1976) on undiluted and diluted samples within 15 min of semen collection (controls) and after being held at 4 1 , 25, 15, or 5 C for either 3 or 6 hr. One drop of semen was placed on a microscope slide (maintained at room temperature) and examined immediately by phase contrast microscopy (40 x ) . Experiment 3: Measurement of Egg Fer-
1913
CLARKE ET AL.
1914
B
60
^ J
TURKEY 41 C
50
40 J CHICKEN 41 C 30 CHICKEN 41 C
r
r-'^
—T
T-"~r 2
3
4
1 CHICKEN 25 C J TURKEY 25 C
j TURKEY 25 C " * " ] CHICKEN 25 C
> -<
20 TURKEY 41 C 10
5 INCUBATION TIME (hr :
(hr )
.
B
J TURKEY 15 C [ CHICKEN 15 C
•
""-"---v - J
...-J_-
-4
1 - — "T— 2
3
4
INCUBATION TIME (hr )
i—
—-r-
_. j
TURKEY 5 C
— J CHICKEN 5 C
3 4 5 INCUBATION TIME (hr )
PIG. 1. The respiratory rate of undiluted (A) and diluted (B) chicken and turkey sperm incubated at various temperatures. (Mean and SEM of 6 determinations.)
weighted ANOVA, data being weighted by the square root of the number of eggs laid per hen. Differences between treatment means were determined using the Student-Newman-Keuls multiple comparison test in all cases. Pooled standard errors of the mean (SEM) were calculated from the ANOVA. RESULTS
Experiment 1. The respiration of chicken and turkey spermatozoa decreased significantly (P<.05) as the incubation temperature was lowered from 41 to 5 C for all treatments. No oxygen was taken up by sperm when incubated for 3 or 6 hr at 5 C (Table 1). With the exception of diluted chicken semen incubated at 41 C for 3 hr, respiration of chicken and turkey sperm in diluted semen was significantly (P<.05) higher than in undiluted semen incubated under similar conditions. The cumulative oxygen consumption by spermatozoa in undiluted chicken or turkey semen ceased
after 3 hr of incubation at 15 C but increased significantly between 3 and 6 hr of incubation at 41 o r 2 5 C . With undiluted semen, chicken sperm respired at a rate higher than undiluted turkey sperm when incubated at 41 C under similar conditions, but there was no difference at 25 or 15 C (Fig. la). With diluted semen, turkey sperm respired at a higher rate than chicken sperm after 1 hr of incubation at 41 C, but no differences were observed at 25 or 15 C (Fig. lb). Experiment 2. The percentage of progressively motile chicken and turkey sperm held at 41 C was significantly (P<.05) lower than the value for controls and for those held at 25, 15, or 5 C (Table 2). With the exception of undiluted chicken semen held for 6 hr at 15 C and 5 C, sperm motility of samples held at these temperatures did not differ significantly (P>.05) from that of the unstored controls. At 25 C, diluted chicken semen held for 3 hr was the
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INCUBATION TIME
HOLDING CHICKEN AND TURKEY SEMEN
1915
TABLE 2. Percent ofprogessively motile chicken and turkey spermatozoa held at various temperatures and incubation times
Inculbation time (hr)
Semen Chicken Undiluted Diluted Turkey Undiluted
3 6 3 6
Control
41 C
25 C
84.3 a . e
12.1 c . e . f 7 d,f 25.0b,e 17.8c>e 9.3 c > e . f .7Cf 14.3 c . e 5.0C,e,f
87.1 a . e
93.6 a . e 93.6 a ' e
ISC
5C
57.8 b . f .g 42.2 c .g 77.1a.e 65.7 b . e .g
77.8 a . e 65.0b,e 82.8 a . e 77.8 a . e
80.0 a . e 70.0 b ,e 87.9 a . e 83.6 a > e
80.0 b . e 74.3 b . e 60.7 b > f 41.4 b .g
89.3 a . b > e 88.6 a . e 92.1 a - e 90.0 a . e
92.1 a - e 87.1 a > e 92.1a.e 89.3 a > e
' ' ' Means with different superscripts within rows plus control differ significantly (P<.05). ans with different superscripts within columns differ significantly (P<.05). 1
All values are means of 15 determinations (pooled SEM = ± 5.556).
only sample t h a t did n o t differ from t h e controls. Sperm motility of neither species was affected b y semen dilution when i n c u b a t e d at 15 and 5 C. A higher level of chicken sperm motility was n o t e d w h e n diluted and stored at 25 C, whereas t u r k e y sperm held at this temperature was n o t affected by semen dilution. Sperm motility of t h e c o n t r o l semen samples from b o t h species was unaffected b y dilution. There was a significant ( P < . 0 5 ) species-dilution
interaction with respect t o sperm m o t i l i t y . Experiment 3. T h e fertility of u n d i l u t e d chicken semen held for 3 (41%) and 6 h r (28%) at 5 C was higher t h a n t h a t of samples held at higher t e m p e r a t u r e s b u t significantly l o w e r t h a n t h e u n s t o r e d control ( 8 5 % ; T a b l e 3). Regardless of die holding t e m p e r a t u r e , t h e fertilizing capacity of u n d i l u t e d chicken semen was severely diminished after 3 and 6 hr. T h e fertility of diluted chicken semen held for 3 h r at 2 5 , 15, and 5 C was n o t significantly dif-
TABLE 3. Percent weighted fertility of chicken and turkey sperm held at various temperatures and incubation times
Incubation time (hr)
Semen Chicken Undiluted Diluted Turkey Undiluted Diluted
Percent weighted fertility 1 Control
3 6 3 6
85.0 a . e
3 6 3 6
80.0 a . e
81.0 a . e
68.0a>f
41 C
Od.f 0 d,f 2ld,e 3d,f ld,e ld,e 0 d ,e 0 d ,e
25 C
15C
10 d ,c,g l d ,g 79a'e 61c-f
18 c .g 6 d,h 83 a - e 6 4 b,c,f
4 1 b,f c
82 a > c 56 b ,f 45 b .g 12d,h
68 b > e 55b,f 20 c .g ld,h
21c.f 39 c ' e 29 d ,c,f 24C.f
a,b,c,d Means with different superscripts within rows plus control differ significantly (P<.05). e,f,g,h 1
Means with different superscripts within columns differ significantly (P<.05).
All values are means of 7 weekly determinations (pooled SEM = + 4.497).
5C
28 >g 71a-e 75a.b.e
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Diluted
3 6 3 6
Percent progressive motility 1
CLARKE ET AL.
1916
DISCUSSION
That semen dilution increased chicken and turkey sperm respiration was not surprising and was probably due to an increased availability of nutrients, such as fructose, or was increased by dilution per se, The BPSE contains 5 mg/ml of fructose, a level 100 times greater than that found in undiluted semen (Kamar and Rizik, 1972). Thus, the added fructose from BPSE could account for the higher respiratory activity. Furthermore, semen dilution has been shown to stimulate sperm metabolism (Sexton, 1976), and this phenomenon has been related to an increase in enzyme activity associated with the tricarboxylic acid (TCA) cycle (Smith etal, 1957). In diluted semen, the respiratory rate of turkey sperm was higher than that of chicken sperm at 41 C, which is in contradiction to earlier reports comparing the respiration of washed chicken and turkey spermatozoa. Sexton (1974) found washed turkey sperm to have lower endogenous and exogenous (as determined by the addition of sugar and amino acids) respiratory rates than chicken sperm when incubated at 41 C. One possible explanation for the conflicting results is that the respiratory rate of turkey sperm may be
negatively influenced by the technique of washing. That turkey sperm are more fragile than chicken sperm is quite evident from the data in Table 3. Three independent trials were performed in an attempt to establish the cause of the negative respiratory readings at 5 C incubation. In the first trial, addition of antibacterials (see Sexton et ah, 1980) to the incubation medium did not have a significant effect on respiration, which indicates that oxygenproducing bacteria were not a factor. The influence of chemical breakdown products formed from extender solutes and the water solvent was studied in Trial 2. Heat treatment was used to accelerate chemical decomposition. Chemical degradations were not responsible for the negative readings. In Trial 3, a comparison of respirometers was made at 25 C by incubating vessels containing only BPSE. Both respirometers performed identically under these conditions, ruling out the possibility of respirometer malfunction. Therefore, the question of what caused the negative respiration readings at 5 C remains unanswered. It has generally been thought that a reduction in sperm metabolism is required to hold semen in vitro successfully without seriously decreasing its fertilizing ability. This concept appears to be true for chicken semen but not for turkey semen. Both undiluted and diluted turkey semen held at 15 or 5 C exhibited low fertility even though sperm respiratory activity was minimized. Perhaps factors other than low metabolic activity are needed for successful preservation of turkey semen. Semen dilution increased chicken sperm motility, whereas dilution per^ se had little influence on the level of motility of turkey sperm. This indicated that there were rather distinct differences in die effects of BPSE or dilution on the motility of chicken and turkey sperm held at low temperatures. It appears that the motilities of chicken and turkey sperm were stimulated when semen was diluted with BPSE and held at higher temperatures (41 and 25 C). This trend was also evident with chicken sperm held at the lower temperatures (15 and 5 C). However, semen dilution did not affect the motility of turkey sperm at 15 or 5 C. The low fertilizing capacity of diluted turkey sperm, both in the unstored control and treated groups, could have been related to either the extender, holding conditions, or
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ferent from that of the unstored control. There was no decrease in the fertility of diluted chicken semen held at 5 C for 6 hr when compared to the control. At all other holding temperatures, fertility levels were lower than those of the control. The fertilizing capacities of the unstored diluted (81%) and undiluted (85%) controls for chicken semen did not differ. Conversely, die fertilizing capacity of the diluted control turkey semen was significantly lower (68%) than that of the undiluted control (80%). The fertility of diluted turkey semen held at 25 and 15 C for 3 or 6 hr was significantly lower than the fertility of undiluted samples held under similar conditions. Chicken and turkey sperm held at 41 C showed consistently lower fertility levels than sperm held at the lower temperatures. The fertility of diluted and undiluted turkey semen held at 5 C for 3 or 6 hr was lower than that of the unstored controls. There were significant (P<.05) interactions of species-dilution, species-temperature, and temperature-holding time when the fertility data were analyzed.
HOLDING CHICKEN AND TURKEY SEMEN b o t h . It m a y be t h a t one or m o r e c o n s t i t u e n t s of t h e e x t e n d e r failed t o provide a suitable e n v i r o n m e n t to maintain o p t i m u m fertility levels for t u r k e y sperm. T h e dilution effect, which was characterized by an increase in respiratory rate of t u r k e y sperm (Table 1), could have c o n t r i b u t e d t o t h e decrease in fertility (Table 3) as has been reported with m a m m a l i a n sperm (for review see Sexton, 1979). T h u s , it remains uncertain as t o w h e t h e r BPSE or t h e dilution rate of 1:5 caused t h e decline in t h e fertility of t u r k e y semen.
T h e a u t h o r s wish t o t h a n k D. M. Gavelek for her technical assistance and L. Douglass for his statistical advice.
REFERENCES Ashizawa, K., and H. Nishiyama, 1978. Effects of temperature on the vigour of motility, oxygen consumption, and duration of motility of fowl spermatozoa under aerobic conditions. Japanese Poultry Sci. 14:264-266. Bakst, M. R., and T. J. Sexton, 1979. Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing. J. Reprod. Fert. 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. Cecil, H. C , 1981. Effects of dietary protein on body weight and reproductive performance of male turkeys. Poultry Sci. 1049-1055. Cherms, F. L., 1968. Variations in semen quality and the relationship of semen quality to fertility in turkeys. Poultry Sci. 47:746-754. 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. Kamar, G. A., and M. A. Risik, 1972. Semen characteristics of two breeds of turkeys. J. Reprod. Fertil. 29:317-325. Lake, P. E., 1971. Physiology and biochemistry of poultry semen. Pages 93—128 in Advances in Reproductive Physiology. M.W.H. Bishop, ed. Academic Press, New York, NY. Litjen, J. B., 1972. Artificial insemination in turkey and an investigation into a suitable diluent for turkey semen. 8th Int. Congr. Anim. Reprod. Artif. Insem. 3:1698-1701. Sexton, T. J., 1974. Oxidative and glycolytic activity of chicken and turkey spermatozoa. Comp. Biochem. Physiol. 48B:59-65. Sexton, T. J., 1976. Studies on the dilution of turkey semen. Br. Poultry Sci. 17:179-186. Sexton, T. J., 1978. A new poultry semen extender. 3. Effect of storage conditions on the fertilizing capacity of chicken semen stored at 5 C. Poultry Sci. 47:285-289. Sexton, T. J., 1979. Preservation of poultry semen. A review. Pages 159—170 in Beltsville Symposium in Agricultural Research. 3. Animal Reproduction. Allanheld, Osmun & Co., Montclair, NJ. Sexton, T. J., L. A. Jacobs, and G. R. McDaniel, 1980. A new poultry semen extender. 4. Effects of antibacterials in control of bacterial contamination in chicken semen. Poultry Sci. 59: 274-281. Smith, J. T., D. T. Mayer, and C. P. Merilan, 1957. Seasonal variation in the succinic dehydrogenase activity of bovine spermatozoa. J. Dairy Sci. 40:516-520. Van Wambeke, F., 1970. The effect of two insemination methods on the fertilizing capacity of fowl semen stored for 24 and 48 hrs. Pages 349—351 in Proc. 14th World's Poultry Congr. Van Wambeke, F., 1972. Fertility and hatchability results with fowl spermatozoa stored in fresh and freeze-dried diluent. Br. Poultry Sci. 13:179— 183. Winer, B. J., 1971. Statistical Principles in Experimental Design. 2nd ed. McGraw-Hill Book Co., New York, NY.
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ACKNOWLEDGMENTS
1917