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Testicular Weights, Ductus Deferens Semen Volumes, and Sperm Concentration of Turkeys with High and Low Ejaculate Volumes
Testicular Weights, Ductus Deferens Semen Volumes, and Sperm Concentration of Turkeys with High and Low Ejaculate Volumes HELENE C. CECIL and MURRAY R. BAKST US Department of Agriculture, ARS, Avian Physiology Laboratory, Beltsville, Maryland 20705 (Received for publication July 7, 1983)
1984 Poultry Science 63:1432-1437 INTRODUCTION Ejaculate volumes of breeder t u r k e y s can vary from .06 to .8 ml b e t w e e n individuals within a flock, b u t volumes for ejaculates collected once-a-week from the same individual are q u i t e consistent (Cecil, 1 9 8 1 ; K a m m e r e r et al., 1972). A flock m a y have 40% of t h e turkeys with ejaculate volumes less than .25 ml, and a p p r o x i m a t e l y 2 5 % with ejaculate volumes greater t h a n .35 ml (Cecil, 1 9 8 1 ; Miller et al., 1977). Whether such variation is genetic or reflects t h e response of individual males to manual stimulation is n o t k n o w n . High semen producers could have a correspondingly high rate of testicular sperm p r o d u c t i o n , or t h e y could just be m o r e efficient in t h e mobilization of d u c t u s (d.) deferens semen at t h e time of manual stimulation and ejaculation. Although m a t u r e male t u r k e y s can be classified as high (HSP) or low (LSP) semen producers, little is k n o w n of their comparative ability t o p r o d u c e , store, and ejaculate sperm. Within t h e reproductive system, the size or weight of t h e testes is indicative of t h e gonadal mass involved in t h e p r o d u c t i o n of sperm and t h e semen within t h e d. deferens is indicative of t h e a m o u n t of m a t u r e sperm stored for ejaculation. T h e present s t u d y compares t h e testis weights and t h e v o l u m e , sperm c o n c e n t r a t i o n , and total n u m b e r of sperm in t h e ejaculate and
d. deferens semen of male t u r k e y s classified as low or high semen producers.
MATERIALS AND METHODS Large White breeder males (Flock 1, J a n u a r y hatch of female-line breeders; Flock 2, August h a t c h , and Flock 3, F e b r u a r y h a t c h of male-line breeders) were housed in floor pens. At 2 4 weeks of age t h e y were subjected t o 14 hr incandescent light per day with an average intensity of 6.5 lx. Once a week, starting at 23 to 28 weeks of age and continuing t h r o u g h o u t t h e e x p e r i m e n t , semen from 3 to 4 cloacal strokes was collected b y abdominal massage (Burrows and Q u i n n , 1937) into 5-ml conical tubes graduated in .1-ml increments. T h e semen volume for each t u r k e y was estimated and recorded t o t h e nearest .02 ml. T h e c o n c e n t r a t i o n of sperm in each semen sample was measured b y the turbidity m e t h o d (Cecil, 1 9 8 2 ) . T u r k e y s t h a t consistently yielded less t h a n .22 ml of semen were classified as LSP and t h o s e t h a t yielded m o r e than .30 ml were classified as HSP. T h e LSP and HSP were further divided into t w o groups: males in one g r o u p (Ejac) were killed immediately after t h e last semen collection; those in t h e o t h e r g r o u p ( N o t Ejac) were killed 1 week after t h e last semen collection.
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ABSTRACT Studies were conducted to determine if high and low ejaculate volumes were associated with differences in testicular weight and ductus (d.) deferens semen volumes. Mature male breeder turkeys were classified as low (=S.22 ml) or high (>.30 ml) semen producers. One week after or immediately after the last semen collection the turkeys were killed, testes weighed, and d. deferens and ejaculate semen volumes and sperm concentrations determined. Comparisons of low (LSP) and high (HSP) semen producers showed no differences in testes weights and sperm concentration in d. deferens. However, LSP turkeys had a lower volume of semen in the d. deferens and ejaculated a lower percentage of that volume than did HSP turkeys (P<.05). Although the sperm concentrations of HSP and LSP were the same, the total number of sperm in the ejaculate of LSP was less than the HSP and was also less when calculated as a percentage of the sperm in the ejaculate plus d. deferens. These studies indicate that both a decreased reservoir of semen in the d. deferens and a lower efficiency of removal of semen from the d. deferens contribute to the decreased ejaculate volume of LSP. (Key words: male turkey, semen, ductus deferens, transparent fluid, airsacculitis)
DUCTUS DEFERENS AND EJACULATE SEMEN VOLUMES
inseminated with an aliquot of semen containing 200 X 10 6 sperm. Eggs were collected daily and set weekly for 7 weeks. On Day 7 of incubation the eggs were candled for viable embryos, and the percent fertility was expressed as the percent candling fertility. On Day 28, percent hatchability was determined. In the male line flocks, 17 turkeys had cystic abdominal air sacs, which were presumed to be airsacculitis. The data for the turkeys with airsacculitis are presented separately from normal turkeys. No other gross pathological lesions were observed. Means for the HSP and LSP were compared statistically using the t test (SAS Staff, 1982). RESULTS AND DISCUSSION
Testes weights from the LSP turkeys were similar to those from the HSP (Table 1), and consequently, the differences in ejaculate volumes between LSP and HSP turkeys cannot be attributed to consistent differences in gonadal mass. The right testis consistently weighed less than the left testis and averaged 33
TABLE 1. Testes weights of Large White male turkeys classified as low (LSP) and high (HSP) semen producers Testis weight Group 1
Flock
Age
n
Right
(weeks) Male-line LSP-Not Ejac HSP-Not Ejac
42-45 42-45
Left •(g)
11 18
Total
Right testis weight (% of total)
—
20.0 14.7* 12.8 16.9
23.6 21.9
43.6 36.6*
45.9 39.7
21.7 28.4
34.5 45.7
36.6 37.8
15.9 18.7
23.6 25.3
39.5 44.0
40.6 43.3
15.5 13.5
21.1 23.0
36.7 36.4
42.3 36.2*
14.4 15.6
20.2 22.8
34.6 38.4
40.8 41.3
LSP-Not Ejac HSP-Not Ejac
54 54
LSP-Ejac HSP-Ejac
38-39 38-39
LSP-Ejac HSP-Ejac
42-45 42-45
LSP-Ejac HSP-Ejac
46-48 ^6-48
LSP-Ejac HSP-Ejac
54 54
12
8.6 11.8*
15.2 16.8
23.8 28.6
36.6 41.0
54-59 54-59 53-55 53-55
10 7 6 6
7.4 11.1 9.4 12.6
13.6 17.8
21.0 28.8
33.3 38.3
14.9 18.8
24.2 31.4
38.4 40.1
Female-line LSP-Not Ejac HSP-Not Ejac LSP-Ejac HSP-Ejac
1 1 1 1
11 5 11 11
1 LSP = ejaculate volumes <.22 ml per ejaculate; HSP = ejaculate volumes >.30 ml per ejaculate. Males in Ejac group were killed immediately after the last semen collection; those in the Not Ejac group were killed 1 week after the last semen collection.
*P<.05; t test compared HSP and LSP in each age group.
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Between 53 and 59 weeks of age (29 Flock 1 female-line males) and between 38 and 54 weeks (136 male-line males), LSP and HSP turkeys were killed with sodium pentobarbital. The testes, excurrent duct system (epididymal region and d. deferens), and cloaca were removed in toto. The distal segment of the d. deferens was isolated from the cloaca and the excurrent duct system was then cleaned of connective tissue. The d. deferens was separated from each testis at the epididymal/d. deferens junction and the semen manually expressed from each d. deferens into a graduated test tube (Bakst and Cecil, 1981). The volume and sperm concentration of d. deferens semen were measured as described above for ejaculates. The right and left testes, including the epididymal region, were weighed. Each week for 3 weeks the ejaculate and d. deferens semen from 3 individual males in the HSP and LSP (Ejac, 52 to 54-week-old male line) were diluted 1:2 with Beltsville Poultry Semen Extender (Sexton, 1980) and 3 hens per diluted semen sample (9 hens/group) were each
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42-45 42-45
46-48 46-48
54 54
LSP-Ejac HSP-Ejac
LSP-Ejac HSP-Ejac
LSP-Ejac HSP-Ejac
10 7
6 6
1 1
1 1
* *
.18 34**
A.A.* * * *
.16 18 46***
15 £. 1 * * * *
C£*** *
16
18 52* * * *
15 52****
Actual 2
(-Q* * * *
Ejaculate
.16
.16 .56****
40* * #*
.17
AC**
.13
.18 4<5** * *
.12 .40****
Previous 1 . ..
Actual ejaculate volume = the ejaculate volume immediately before d. deferens removal.
Previous ejaculate volume = the mean ejaculate volume for 5 previous weeks.
53-55 53-55
9 8
3 3
6 12
11 11
3 3
2 2
5 8
9 11
11 18
n
3 3
3 3
3 3
Flock
.21 .31 .21
.21 .27 .24 .26
.25 .38 .15 .18
.13 .13
.14 .30**
.36 .45
.15 .38**
.11 .18
.26 .43*
.35 .54
.33 .52*
2 C* * *
.32 .52***
.41 .49
fmll
(mi)
Left
d. deferens Right
*P«.05; **P«.01; ***P«.001; ****P«.0001; t test compared HSP and LSP in each age group.
2
1
LSP-Ejac HSP-Ejac
54-59 54-59
38-39 38-39
LSP-Ejac HSP-Ejac
Female-line LSP-Not Ejac HSP-Not Ejac
54 54
42-45 42-45
(weeks)
Age
LSP-Not Ejac HSP-Not Ejac
Male-line LSP-Not Ejac HSP-Not Ejac
Group
TABLE 2. Ejaculate and ductus (d.) deferens semen volumes of Large White male turkeys classified as low (LSP) and volumes were measured immediately after semen collection (Ejac) or 1 week after the last seme
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D U C T U S D E F E R E N S A N D E J A C U L A T E SEMEN V O L U M E S
T A B L E 3. Sperm concentration of ejaculate and ductus (d.) deferens semen of Large White male turkeys classified as low (LSP) and high (HSP) semen producers. Ductus deferens sperm concentrations were measured immediately after semen collection (Ejac) or 1 week after the last semen collection (Not Ejac) R a t i o of ejaculate t o Avg d.deferens
d. deferens Age
Group
Ejaculate
n
Flock
Left
Avg
(sperm X
(weeks) 3 3
11 18
9.7 9.5
9.8 9.9
9.7 9.7
LSP-Not Ejac 54 HSP-Not Ejac 54
3 3
9 11
8.1 9.1
8.6 9.0
8.5 9.0
LSP-Ejac HSP-Ejac
38-39 38-39
3 3
5 8
9.6 10.0
10.4 10.0
10.0 10.0
LSP-Ejac HSP-Ejac
42-45 42-45
3 3
11 11
6.9 8.1
8.9 9.3
LSP-Ejac HSP-Ejac
46-48 46-48
3 3
9 8
6.6 8.5
9.9 10.0
LSP-Ejac HSP-Ejac
54 54
2 2
6 12
7.0 8.6
1 1
8 7
1 1
6 6
Female-line LSP-Not Ejac 54-59 HSP-Not Ejac 54-59 LSP-Ejac HSP-Ejac
53-55 53-55
*P<.05i * * P « . 0 1 ; * * * P « . 0 0 1 ; '
T A B L E 4. Contribution
7.1 9.2**
9.4 9.3
72 91**
8.7 9.7
79 84
10.3 10.3
10.2 10.2
66 87*
9.0 8.9
8.4 9.4
9.0 9.3
85 94
16.4 17.9
17.7 16.3
17.7 17.2
13.6 17.0
10.3 16.6
10.9 16.5
8.5 9.9*
.89 .67
* P < . 0 0 0 1 ; t test compared HSP and LSP in each age g r o u p .
of ductus
(d.) deferens
semen
(DD) and transparent
fluid
Ejaculate
DD2
(TF) to
ejaculate
Volume from Group1
Flock
Age (weeks)
\uu)
TF3
—
-
Male line LSP-Ejac HSP-Ejac
38-39 38-39
.15 59** * *
.11 4 7 * * **
.04 .05
LSP-Ejac HSP-Ejac
42-45 42-45
.18 52* * * *
.14 4 3 * * **
.04 .09
LSP-Ejac HSP-Ejac
46-48 46-48
.16 56****
.11 54***
.04 .06
LSP-Ejac HSP-Ejac
54 54
.15 .61****
.13 .57***
.03 .03
1 T u r k e y s were classified as low (LSP) or high (HSP) semen p r o d u c e r s . T h e d. deferens sperm c o n c e n t r a t i o n s were measured immediately after semen collection (Ejac). 2 Milliliters semen from d. deferens = total n u m b e r of sperm in ejaculate divided by the n u m b e r of sperm per milliliters of d. deferens semen.
'Milliliters transparent fluid = milliliters ejaculate minus the calculated milliliters semen from d. deferens. * * * P « . 0 0 1 ; * * * * P « . 0 0 0 1 ; t test c o m p a r e d HSP and LSP in each age g r o u p .
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Male-line LSP-Not Ejac 42-45 HSP-Not Ejac 42-45
Right
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CECIL AND BAKST ejaculate volumes of HSP when compared to the LSP. Although the sperm concentrations of the ejaculate of HSP and LSP were similar (Table 3), the ejaculate sperm concentrations were consistently lower than the d. deferens sperm concentrations. When the ejaculate is manually collected, it is diluted with transparent fluid, a lymph-like fluid expressed from the plicae lymphaticae (lymph folds). We assume that the lowered sperm concentration in the ejaculate is due to the addition of transparent fluid. Calculations (described in footnotes, Table 4) made from comparisons of the volume and total sperm number in the ejaculate with the sperm concentration in the d. deferens show that the LSP turkeys (Table 4) had .15 to .18 ml ejaculate with .11 to .14 ml from the d. deferens and .03 to .04 ml transparent fluid; HSP turkeys had .52 to .61 ml ejaculate with .43 to .57 ml from the d. deferens and .03 to
TABLE 5. Total sperm in ejaculate and ductus (d.) deferens semen of Large White male turkeys classified as low (LSP) and high (HSP) semen producers. Ductus deferens total sperm were measured immediately after semen collection (Ejac) or 1 week after the last semen collection (Not Ejac) d. cdeferens Group
Age
Flock
n
Ejaculate -
(weeks)
Left
Right (sperm
w
Total1
1 f\9 \
(% of total)
Male-line LSP-Not Ejac 42-45 HSP-Not Ejac 42-45
3 3
11 18
3.9 4.6
3.1 5.2**
7.0 97**
LSP-Not Ejac 53-55 HSP-Not Ejac 53-55
3 3
9 11
2.8 4.8*
3.1 4.8
5.9 9.6*
LSP-Ejac HSP-Ejac
38-39 38-39
3 3
5 8
A y* * * *
2.0 2.7
2.2 3.1
LSP-Ejac HSP-Ejac
42-45 42-45
3 3
11 11
A 9* * * *
2.2 2.5
5.3 1.8 3.5*** 10.2****
LSP-Ejac HSP-Ejac
46-48 46-48
3 3
9 8
1.1 5.0***
2.7 4.4
3.8 5.1
LSP-Ejac HSP-Ejac
54 54
2 2
6 12
1.1 5.3***
1.3 1.7
1.4 2.9*
1 1
8 7
3.1 6.6**
4.5 5.9
1 1
6 6
1.6 1.6
1.6 2.4
Female-line LSP-Not Ejac 54-59 HSP-Not Ejac 54-59 LSP-Ejac HSP-Ejac
53-55 53-55
1.1 1.2
1.5 4.4**
Ejaculate
5.3 10.5***
7.7 15.2*** 4.1 9.8**
23 45*** 26 42*** 19 35* 34 54**
7.8 12.5 4.6 8.4**
33 51*
1 Not Ejac total = right d. deferens plus left d. deferens; Ejac total = ejaculate plus right d. deferens plus left d. deferens.
* P < . 0 5 ; * * P < . 0 1 ; * * * P « . 0 0 1 ; * * * * P « . 0 0 0 1 ; t test c o m p a r e d HSP and LSP in each age g r o u p .
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to 46% of the total testes weight. Of the 119 male-line turkeys in this study > 112 had aright testis weight less than the left testis weight (paired t test, P<.05). In both male and female lines the sum of the ejaculate and d. deferens semen volume for turkeys with a semen collection immediately before sacrifice (Ejac, Table 2) and the semen volume in the d. deferens 1 week after the last semen collection (Not Ejac, Table 2) was greater for HSP than LSP. In addition, a larger percentage of the total available semen volume (d. deferens + ejaculate) appeared in the ejaculate of HSP (Table 2, actual ejaculate, 40 to 61% of total volume) than in the ejaculate of LSP (Table 2, actual ejaculate, 24 to 40% of total volume). Therefore, it appears that both a greater reservoir of semen in the d. deferens and a greater efficiency of removal of semen from the d. deferens, rather than a greater testes weight, contribute to the larger
DUCTUS DEFERENS AND EJACULATE SEMEN VOLUMES TABLE 6. Relationship of ejaculate volumes to ductus (d.) deferens volumes in turkeys with airsacculitis Not ejaculated (n = 12)
.15 ... .75 .75
Sperm concentration, Sperm X 10'/ml Ejaculate ... d. deferens 9.5 Total sperm, sperm X 10 9 Ejaculate .. d. deferens 7.48 2 Total semen 7.48
.12 .12 .63 .75 5.4 10.4 .78 6.61 7.39
1 Previous ejaculate volume = the mean ejaculate volume for 5 previous weeks. 2
Total semen = ejaculate plus d. deferens.
.09 ml transparent fluid. Therefore, transparent fluid cannot be considered a source of the greater ejaculate volumes of the HSP. In all treatment groups the total sperm number in d. deferens semen was greater for HSP than LSP (Table 5). Likewise, a larger percentage of the d. deferens sperm appeared in the ejaculate. The differences in total sperm numbers between the HSP and LSP can be attributed to the greater volumes in HSP (Table 2) as the sperm concentrations (Table 3) were generally similar for HSP and LSP. During Week 7 (the 4th week after the last insemination) fertility of eggs set was 79, 88, 88, and 81%, and hatch of fertile eggs was 95, 89, 86, and 95% for eggs from hens inseminated with HSP ejaculate, LSP ejaculate, HSP d. deferens semen, and LSP d. deferens semen, respectively. Overall fertility for the 7 weeks was 90, 93, 94, and 90% and overall hatch of fertile eggs was 89, 90, 90, and 95% for HSP ejaculate, LSP ejaculate, HSP d. deferens semen, and LSP d. deferens semen, respectively. These data demonstrate no differences in fertility or hatchability after inseminations of comparable numbers of sperm from HSP and LSP. Table 6 shows the data for 17 males with airsacculitis, 16 of which had been classified as LSP. The d. deferens volume of the turkeys with airsacculitis was .75 ml and the ejaculate contained 17% of the d. deferens semen volume and 11% of the d. deferens sperm, values that
are similar to those of normal LSP turkeys. However, the classification of 16 of the 17 turkeys with airsacculitis as LSP indicates that the disease probably lowered the d. deferens volume, as well as the efficiency of removal of the d. deferens semen at ejaculation. Also, the ejaculate of airsacculitis turkeys was diluted to a greater extent with transparent fluid (42%) than that of normal turkeys (13 to 29%). This study has shown that turkeys with ejaculate volumes greater than .30 ml have a greater volume of semen in the d. deferens and are also more efficient in the removal of d. deferens semen at ejaculation than turkeys with ejaculate volumes of less than .22 ml of semen. However, more than half of the volume of semen remains in the d. deferens after ejaculation. The physiological mechanisms that control semen storage and ejaculation in the turkey have yet to be elucidated. A better understanding of the fundamental principles controlling semen production and ejaculation may lead to an increase in semen production and indicate ways to improve semen collection techniques. ACKNOWLEDGMENTS The authors wish to thank D. Bailay and A. Kimrey for technical assistance; W. Nye and S. Greene for semen collection; B. Weinland for assistance with the statistical analysis; and J. Tishue for typing the manuscript. REFERENCES Bakst, M. R., and H. C. Cecil, 1981. Changes in the characteristics of turkey ejaculated semen and ductus deferens semen with repeated ejaculations. Reprod. Nutr. Dev. 21:1095-1103. Burrows, W. H.,and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16:19-24. Cecil, H. C., 1981. Variability in the onset of semen production, semen volume, and sperm concentration in male turkeys. Poultry Sci. 60:1637. (Abstr.) Cecil, H. C , 1982. Effects of frequency of semen collection on reproductive performance of male turkeys fed low protein diets during the breeder period. Poultry Sci. 61:1866-1872. Kammerer, D. M., R. E. Moreng, H. D. Mueller, and H. W. Hobbs, 1972. Turkey semen evaluation for fertility prediction. Poultry Sci. 51:77—82. Miller, D. H., J. W. Bradley, and T. M. Ferguson, 1977. Effect of vitamin A and protein levels on growth and semen quality of turkeys. Poultry Sci. 56:1086-1091. Sexton, T. J., 1980. A new poultry extender. 5. Relationship of diluent components to cytotoxic effects of dimethylsulfoxide on turkey spermatozoa. Poultry Sci. 59:1142-1144. SAS Staff, 1982. SAS User's Guide. SAS Inst., Inc., Cary, NC.
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Semen volume, ml Previous ejaculate1 Actual ejaculate d. deferens Total semen3
Ejaculated (n = 5)
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1984 Poultry Science 63:1432-1437 INTRODUCTION Ejaculate volumes of breeder t u r k e y s can vary from .06 to .8 ml b e t w e e n individuals within a flock, b u t volumes for ejaculates collected once-a-week from the same individual are q u i t e consistent (Cecil, 1 9 8 1 ; K a m m e r e r et al., 1972). A flock m a y have 40% of t h e turkeys with ejaculate volumes less than .25 ml, and a p p r o x i m a t e l y 2 5 % with ejaculate volumes greater t h a n .35 ml (Cecil, 1 9 8 1 ; Miller et al., 1977). Whether such variation is genetic or reflects t h e response of individual males to manual stimulation is n o t k n o w n . High semen producers could have a correspondingly high rate of testicular sperm p r o d u c t i o n , or t h e y could just be m o r e efficient in t h e mobilization of d u c t u s (d.) deferens semen at t h e time of manual stimulation and ejaculation. Although m a t u r e male t u r k e y s can be classified as high (HSP) or low (LSP) semen producers, little is k n o w n of their comparative ability t o p r o d u c e , store, and ejaculate sperm. Within t h e reproductive system, the size or weight of t h e testes is indicative of t h e gonadal mass involved in t h e p r o d u c t i o n of sperm and t h e semen within t h e d. deferens is indicative of t h e a m o u n t of m a t u r e sperm stored for ejaculation. T h e present s t u d y compares t h e testis weights and t h e v o l u m e , sperm c o n c e n t r a t i o n , and total n u m b e r of sperm in t h e ejaculate and
d. deferens semen of male t u r k e y s classified as low or high semen producers.
MATERIALS AND METHODS Large White breeder males (Flock 1, J a n u a r y hatch of female-line breeders; Flock 2, August h a t c h , and Flock 3, F e b r u a r y h a t c h of male-line breeders) were housed in floor pens. At 2 4 weeks of age t h e y were subjected t o 14 hr incandescent light per day with an average intensity of 6.5 lx. Once a week, starting at 23 to 28 weeks of age and continuing t h r o u g h o u t t h e e x p e r i m e n t , semen from 3 to 4 cloacal strokes was collected b y abdominal massage (Burrows and Q u i n n , 1937) into 5-ml conical tubes graduated in .1-ml increments. T h e semen volume for each t u r k e y was estimated and recorded t o t h e nearest .02 ml. T h e c o n c e n t r a t i o n of sperm in each semen sample was measured b y the turbidity m e t h o d (Cecil, 1 9 8 2 ) . T u r k e y s t h a t consistently yielded less t h a n .22 ml of semen were classified as LSP and t h o s e t h a t yielded m o r e than .30 ml were classified as HSP. T h e LSP and HSP were further divided into t w o groups: males in one g r o u p (Ejac) were killed immediately after t h e last semen collection; those in t h e o t h e r g r o u p ( N o t Ejac) were killed 1 week after t h e last semen collection.
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ABSTRACT Studies were conducted to determine if high and low ejaculate volumes were associated with differences in testicular weight and ductus (d.) deferens semen volumes. Mature male breeder turkeys were classified as low (=S.22 ml) or high (>.30 ml) semen producers. One week after or immediately after the last semen collection the turkeys were killed, testes weighed, and d. deferens and ejaculate semen volumes and sperm concentrations determined. Comparisons of low (LSP) and high (HSP) semen producers showed no differences in testes weights and sperm concentration in d. deferens. However, LSP turkeys had a lower volume of semen in the d. deferens and ejaculated a lower percentage of that volume than did HSP turkeys (P<.05). Although the sperm concentrations of HSP and LSP were the same, the total number of sperm in the ejaculate of LSP was less than the HSP and was also less when calculated as a percentage of the sperm in the ejaculate plus d. deferens. These studies indicate that both a decreased reservoir of semen in the d. deferens and a lower efficiency of removal of semen from the d. deferens contribute to the decreased ejaculate volume of LSP. (Key words: male turkey, semen, ductus deferens, transparent fluid, airsacculitis)
DUCTUS DEFERENS AND EJACULATE SEMEN VOLUMES
inseminated with an aliquot of semen containing 200 X 10 6 sperm. Eggs were collected daily and set weekly for 7 weeks. On Day 7 of incubation the eggs were candled for viable embryos, and the percent fertility was expressed as the percent candling fertility. On Day 28, percent hatchability was determined. In the male line flocks, 17 turkeys had cystic abdominal air sacs, which were presumed to be airsacculitis. The data for the turkeys with airsacculitis are presented separately from normal turkeys. No other gross pathological lesions were observed. Means for the HSP and LSP were compared statistically using the t test (SAS Staff, 1982). RESULTS AND DISCUSSION
Testes weights from the LSP turkeys were similar to those from the HSP (Table 1), and consequently, the differences in ejaculate volumes between LSP and HSP turkeys cannot be attributed to consistent differences in gonadal mass. The right testis consistently weighed less than the left testis and averaged 33
TABLE 1. Testes weights of Large White male turkeys classified as low (LSP) and high (HSP) semen producers Testis weight Group 1
Flock
Age
n
Right
(weeks) Male-line LSP-Not Ejac HSP-Not Ejac
42-45 42-45
Left •(g)
11 18
Total
Right testis weight (% of total)
—
20.0 14.7* 12.8 16.9
23.6 21.9
43.6 36.6*
45.9 39.7
21.7 28.4
34.5 45.7
36.6 37.8
15.9 18.7
23.6 25.3
39.5 44.0
40.6 43.3
15.5 13.5
21.1 23.0
36.7 36.4
42.3 36.2*
14.4 15.6
20.2 22.8
34.6 38.4
40.8 41.3
LSP-Not Ejac HSP-Not Ejac
54 54
LSP-Ejac HSP-Ejac
38-39 38-39
LSP-Ejac HSP-Ejac
42-45 42-45
LSP-Ejac HSP-Ejac
46-48 ^6-48
LSP-Ejac HSP-Ejac
54 54
12
8.6 11.8*
15.2 16.8
23.8 28.6
36.6 41.0
54-59 54-59 53-55 53-55
10 7 6 6
7.4 11.1 9.4 12.6
13.6 17.8
21.0 28.8
33.3 38.3
14.9 18.8
24.2 31.4
38.4 40.1
Female-line LSP-Not Ejac HSP-Not Ejac LSP-Ejac HSP-Ejac
1 1 1 1
11 5 11 11
1 LSP = ejaculate volumes <.22 ml per ejaculate; HSP = ejaculate volumes >.30 ml per ejaculate. Males in Ejac group were killed immediately after the last semen collection; those in the Not Ejac group were killed 1 week after the last semen collection.
*P<.05; t test compared HSP and LSP in each age group.
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Between 53 and 59 weeks of age (29 Flock 1 female-line males) and between 38 and 54 weeks (136 male-line males), LSP and HSP turkeys were killed with sodium pentobarbital. The testes, excurrent duct system (epididymal region and d. deferens), and cloaca were removed in toto. The distal segment of the d. deferens was isolated from the cloaca and the excurrent duct system was then cleaned of connective tissue. The d. deferens was separated from each testis at the epididymal/d. deferens junction and the semen manually expressed from each d. deferens into a graduated test tube (Bakst and Cecil, 1981). The volume and sperm concentration of d. deferens semen were measured as described above for ejaculates. The right and left testes, including the epididymal region, were weighed. Each week for 3 weeks the ejaculate and d. deferens semen from 3 individual males in the HSP and LSP (Ejac, 52 to 54-week-old male line) were diluted 1:2 with Beltsville Poultry Semen Extender (Sexton, 1980) and 3 hens per diluted semen sample (9 hens/group) were each
1433
42-45 42-45
46-48 46-48
54 54
LSP-Ejac HSP-Ejac
LSP-Ejac HSP-Ejac
LSP-Ejac HSP-Ejac
10 7
6 6
1 1
1 1
* *
.18 34**
A.A.* * * *
.16 18 46***
15 £. 1 * * * *
C£*** *
16
18 52* * * *
15 52****
Actual 2
(-Q* * * *
Ejaculate
.16
.16 .56****
40* * #*
.17
AC**
.13
.18 4<5** * *
.12 .40****
Previous 1 . ..
Actual ejaculate volume = the ejaculate volume immediately before d. deferens removal.
Previous ejaculate volume = the mean ejaculate volume for 5 previous weeks.
53-55 53-55
9 8
3 3
6 12
11 11
3 3
2 2
5 8
9 11
11 18
n
3 3
3 3
3 3
Flock
.21 .31 .21
.21 .27 .24 .26
.25 .38 .15 .18
.13 .13
.14 .30**
.36 .45
.15 .38**
.11 .18
.26 .43*
.35 .54
.33 .52*
2 C* * *
.32 .52***
.41 .49
fmll
(mi)
Left
d. deferens Right
*P«.05; **P«.01; ***P«.001; ****P«.0001; t test compared HSP and LSP in each age group.
2
1
LSP-Ejac HSP-Ejac
54-59 54-59
38-39 38-39
LSP-Ejac HSP-Ejac
Female-line LSP-Not Ejac HSP-Not Ejac
54 54
42-45 42-45
(weeks)
Age
LSP-Not Ejac HSP-Not Ejac
Male-line LSP-Not Ejac HSP-Not Ejac
Group
TABLE 2. Ejaculate and ductus (d.) deferens semen volumes of Large White male turkeys classified as low (LSP) and volumes were measured immediately after semen collection (Ejac) or 1 week after the last seme
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1435
D U C T U S D E F E R E N S A N D E J A C U L A T E SEMEN V O L U M E S
T A B L E 3. Sperm concentration of ejaculate and ductus (d.) deferens semen of Large White male turkeys classified as low (LSP) and high (HSP) semen producers. Ductus deferens sperm concentrations were measured immediately after semen collection (Ejac) or 1 week after the last semen collection (Not Ejac) R a t i o of ejaculate t o Avg d.deferens
d. deferens Age
Group
Ejaculate
n
Flock
Left
Avg
(sperm X
(weeks) 3 3
11 18
9.7 9.5
9.8 9.9
9.7 9.7
LSP-Not Ejac 54 HSP-Not Ejac 54
3 3
9 11
8.1 9.1
8.6 9.0
8.5 9.0
LSP-Ejac HSP-Ejac
38-39 38-39
3 3
5 8
9.6 10.0
10.4 10.0
10.0 10.0
LSP-Ejac HSP-Ejac
42-45 42-45
3 3
11 11
6.9 8.1
8.9 9.3
LSP-Ejac HSP-Ejac
46-48 46-48
3 3
9 8
6.6 8.5
9.9 10.0
LSP-Ejac HSP-Ejac
54 54
2 2
6 12
7.0 8.6
1 1
8 7
1 1
6 6
Female-line LSP-Not Ejac 54-59 HSP-Not Ejac 54-59 LSP-Ejac HSP-Ejac
53-55 53-55
*P<.05i * * P « . 0 1 ; * * * P « . 0 0 1 ; '
T A B L E 4. Contribution
7.1 9.2**
9.4 9.3
72 91**
8.7 9.7
79 84
10.3 10.3
10.2 10.2
66 87*
9.0 8.9
8.4 9.4
9.0 9.3
85 94
16.4 17.9
17.7 16.3
17.7 17.2
13.6 17.0
10.3 16.6
10.9 16.5
8.5 9.9*
.89 .67
* P < . 0 0 0 1 ; t test compared HSP and LSP in each age g r o u p .
of ductus
(d.) deferens
semen
(DD) and transparent
fluid
Ejaculate
DD2
(TF) to
ejaculate
Volume from Group1
Flock
Age (weeks)
\uu)
TF3
—
-
Male line LSP-Ejac HSP-Ejac
38-39 38-39
.15 59** * *
.11 4 7 * * **
.04 .05
LSP-Ejac HSP-Ejac
42-45 42-45
.18 52* * * *
.14 4 3 * * **
.04 .09
LSP-Ejac HSP-Ejac
46-48 46-48
.16 56****
.11 54***
.04 .06
LSP-Ejac HSP-Ejac
54 54
.15 .61****
.13 .57***
.03 .03
1 T u r k e y s were classified as low (LSP) or high (HSP) semen p r o d u c e r s . T h e d. deferens sperm c o n c e n t r a t i o n s were measured immediately after semen collection (Ejac). 2 Milliliters semen from d. deferens = total n u m b e r of sperm in ejaculate divided by the n u m b e r of sperm per milliliters of d. deferens semen.
'Milliliters transparent fluid = milliliters ejaculate minus the calculated milliliters semen from d. deferens. * * * P « . 0 0 1 ; * * * * P « . 0 0 0 1 ; t test c o m p a r e d HSP and LSP in each age g r o u p .
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Male-line LSP-Not Ejac 42-45 HSP-Not Ejac 42-45
Right
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CECIL AND BAKST ejaculate volumes of HSP when compared to the LSP. Although the sperm concentrations of the ejaculate of HSP and LSP were similar (Table 3), the ejaculate sperm concentrations were consistently lower than the d. deferens sperm concentrations. When the ejaculate is manually collected, it is diluted with transparent fluid, a lymph-like fluid expressed from the plicae lymphaticae (lymph folds). We assume that the lowered sperm concentration in the ejaculate is due to the addition of transparent fluid. Calculations (described in footnotes, Table 4) made from comparisons of the volume and total sperm number in the ejaculate with the sperm concentration in the d. deferens show that the LSP turkeys (Table 4) had .15 to .18 ml ejaculate with .11 to .14 ml from the d. deferens and .03 to .04 ml transparent fluid; HSP turkeys had .52 to .61 ml ejaculate with .43 to .57 ml from the d. deferens and .03 to
TABLE 5. Total sperm in ejaculate and ductus (d.) deferens semen of Large White male turkeys classified as low (LSP) and high (HSP) semen producers. Ductus deferens total sperm were measured immediately after semen collection (Ejac) or 1 week after the last semen collection (Not Ejac) d. cdeferens Group
Age
Flock
n
Ejaculate -
(weeks)
Left
Right (sperm
w
Total1
1 f\9 \
(% of total)
Male-line LSP-Not Ejac 42-45 HSP-Not Ejac 42-45
3 3
11 18
3.9 4.6
3.1 5.2**
7.0 97**
LSP-Not Ejac 53-55 HSP-Not Ejac 53-55
3 3
9 11
2.8 4.8*
3.1 4.8
5.9 9.6*
LSP-Ejac HSP-Ejac
38-39 38-39
3 3
5 8
A y* * * *
2.0 2.7
2.2 3.1
LSP-Ejac HSP-Ejac
42-45 42-45
3 3
11 11
A 9* * * *
2.2 2.5
5.3 1.8 3.5*** 10.2****
LSP-Ejac HSP-Ejac
46-48 46-48
3 3
9 8
1.1 5.0***
2.7 4.4
3.8 5.1
LSP-Ejac HSP-Ejac
54 54
2 2
6 12
1.1 5.3***
1.3 1.7
1.4 2.9*
1 1
8 7
3.1 6.6**
4.5 5.9
1 1
6 6
1.6 1.6
1.6 2.4
Female-line LSP-Not Ejac 54-59 HSP-Not Ejac 54-59 LSP-Ejac HSP-Ejac
53-55 53-55
1.1 1.2
1.5 4.4**
Ejaculate
5.3 10.5***
7.7 15.2*** 4.1 9.8**
23 45*** 26 42*** 19 35* 34 54**
7.8 12.5 4.6 8.4**
33 51*
1 Not Ejac total = right d. deferens plus left d. deferens; Ejac total = ejaculate plus right d. deferens plus left d. deferens.
* P < . 0 5 ; * * P < . 0 1 ; * * * P « . 0 0 1 ; * * * * P « . 0 0 0 1 ; t test c o m p a r e d HSP and LSP in each age g r o u p .
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to 46% of the total testes weight. Of the 119 male-line turkeys in this study > 112 had aright testis weight less than the left testis weight (paired t test, P<.05). In both male and female lines the sum of the ejaculate and d. deferens semen volume for turkeys with a semen collection immediately before sacrifice (Ejac, Table 2) and the semen volume in the d. deferens 1 week after the last semen collection (Not Ejac, Table 2) was greater for HSP than LSP. In addition, a larger percentage of the total available semen volume (d. deferens + ejaculate) appeared in the ejaculate of HSP (Table 2, actual ejaculate, 40 to 61% of total volume) than in the ejaculate of LSP (Table 2, actual ejaculate, 24 to 40% of total volume). Therefore, it appears that both a greater reservoir of semen in the d. deferens and a greater efficiency of removal of semen from the d. deferens, rather than a greater testes weight, contribute to the larger
DUCTUS DEFERENS AND EJACULATE SEMEN VOLUMES TABLE 6. Relationship of ejaculate volumes to ductus (d.) deferens volumes in turkeys with airsacculitis Not ejaculated (n = 12)
.15 ... .75 .75
Sperm concentration, Sperm X 10'/ml Ejaculate ... d. deferens 9.5 Total sperm, sperm X 10 9 Ejaculate .. d. deferens 7.48 2 Total semen 7.48
.12 .12 .63 .75 5.4 10.4 .78 6.61 7.39
1 Previous ejaculate volume = the mean ejaculate volume for 5 previous weeks. 2
Total semen = ejaculate plus d. deferens.
.09 ml transparent fluid. Therefore, transparent fluid cannot be considered a source of the greater ejaculate volumes of the HSP. In all treatment groups the total sperm number in d. deferens semen was greater for HSP than LSP (Table 5). Likewise, a larger percentage of the d. deferens sperm appeared in the ejaculate. The differences in total sperm numbers between the HSP and LSP can be attributed to the greater volumes in HSP (Table 2) as the sperm concentrations (Table 3) were generally similar for HSP and LSP. During Week 7 (the 4th week after the last insemination) fertility of eggs set was 79, 88, 88, and 81%, and hatch of fertile eggs was 95, 89, 86, and 95% for eggs from hens inseminated with HSP ejaculate, LSP ejaculate, HSP d. deferens semen, and LSP d. deferens semen, respectively. Overall fertility for the 7 weeks was 90, 93, 94, and 90% and overall hatch of fertile eggs was 89, 90, 90, and 95% for HSP ejaculate, LSP ejaculate, HSP d. deferens semen, and LSP d. deferens semen, respectively. These data demonstrate no differences in fertility or hatchability after inseminations of comparable numbers of sperm from HSP and LSP. Table 6 shows the data for 17 males with airsacculitis, 16 of which had been classified as LSP. The d. deferens volume of the turkeys with airsacculitis was .75 ml and the ejaculate contained 17% of the d. deferens semen volume and 11% of the d. deferens sperm, values that
are similar to those of normal LSP turkeys. However, the classification of 16 of the 17 turkeys with airsacculitis as LSP indicates that the disease probably lowered the d. deferens volume, as well as the efficiency of removal of the d. deferens semen at ejaculation. Also, the ejaculate of airsacculitis turkeys was diluted to a greater extent with transparent fluid (42%) than that of normal turkeys (13 to 29%). This study has shown that turkeys with ejaculate volumes greater than .30 ml have a greater volume of semen in the d. deferens and are also more efficient in the removal of d. deferens semen at ejaculation than turkeys with ejaculate volumes of less than .22 ml of semen. However, more than half of the volume of semen remains in the d. deferens after ejaculation. The physiological mechanisms that control semen storage and ejaculation in the turkey have yet to be elucidated. A better understanding of the fundamental principles controlling semen production and ejaculation may lead to an increase in semen production and indicate ways to improve semen collection techniques. ACKNOWLEDGMENTS The authors wish to thank D. Bailay and A. Kimrey for technical assistance; W. Nye and S. Greene for semen collection; B. Weinland for assistance with the statistical analysis; and J. Tishue for typing the manuscript. REFERENCES Bakst, M. R., and H. C. Cecil, 1981. Changes in the characteristics of turkey ejaculated semen and ductus deferens semen with repeated ejaculations. Reprod. Nutr. Dev. 21:1095-1103. Burrows, W. H.,and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16:19-24. Cecil, H. C., 1981. Variability in the onset of semen production, semen volume, and sperm concentration in male turkeys. Poultry Sci. 60:1637. (Abstr.) Cecil, H. C , 1982. Effects of frequency of semen collection on reproductive performance of male turkeys fed low protein diets during the breeder period. Poultry Sci. 61:1866-1872. Kammerer, D. M., R. E. Moreng, H. D. Mueller, and H. W. Hobbs, 1972. Turkey semen evaluation for fertility prediction. Poultry Sci. 51:77—82. Miller, D. H., J. W. Bradley, and T. M. Ferguson, 1977. Effect of vitamin A and protein levels on growth and semen quality of turkeys. Poultry Sci. 56:1086-1091. Sexton, T. J., 1980. A new poultry extender. 5. Relationship of diluent components to cytotoxic effects of dimethylsulfoxide on turkey spermatozoa. Poultry Sci. 59:1142-1144. SAS Staff, 1982. SAS User's Guide. SAS Inst., Inc., Cary, NC.
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Semen volume, ml Previous ejaculate1 Actual ejaculate d. deferens Total semen3
Ejaculated (n = 5)
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