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Semen Quality and the Gene for Rose Comb in the Domestic Fowl1
Semen Quality and the Gene for Rose Comb in the Domestic Fowl1 R. D. CRAWFORD2 AND J. ROBERT SMYTH, JR. Department of Poultry Science, University of Massachusetts, Amherst, Massachusetts (Received for publication May 22, 1964)
T
1
Contribution from Massachusetts Agricultural Experiment Station. 2 Present address: Department of Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan.
tion of a female with semen of two or more males. Literature describing the results obtained by this method has been reviewed by Beatty (1960) and by Hess et al. (1958). Russian proponents of the technique claim that its use results in a variety of beneficial results such as improved motility and viability of spermatozoa, higher conception rate and birth rate, lower mortality of offspring, and more rapid growth. However, these claims have not received general support among Western workers. Literature concerning the relationship between fertility and semen quality in the domestic fowl has been reviewed by Lorenz (1959) and by Kamar (1960). These reviews indicate that the percentage of abnormal sperm, initial motility, metabolic activity, and the percentage of live vs. dead sperm are closely related to fertilizing ability of the semen. However, these measures are usually found to be useful mainly for predicting the fertilizing capacity of individual males; when pooled semen is used, the relationships are not so marked. Two reports (Cooper and Rowell, 1958; McDaniel and Craig, 1959) have indicated that there is no relationship between semen volume and fertility, but Kamar (1960) found a significant positive correlation between these traits using a highly selected group of birds. In the review by Lorenz (1959), it is stated that sperm density has not been shown to account for significant amounts of variation in fertility. Since constant volumes are usually inseminated, a relationship would not normally be expected between sperm concentration and fertility.
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HE low fertility characteristic of White Wyandottes is associated with rose comb and is expressed only by males homozygous (RR) for that comb type (Crawford and Merritt, 1963; Crawford and Smyth, 1964a). Presence of the gene for rose comb in the sperm cell does not decrease fertility of that cell; instead, the low fertility is caused by some deleterious effect of the RR male host body acting on the spermatozoa (Crawford and Smyth, 1964b). To determine whether this effect might be identified in the semen, several experiments have been conducted to compare qualities of semen and seminal plasma of males of RR, Rr, and rr comb genotypes. A possible reason for the low fertility of RR males is that some inadequacy might exist in their seminal fluids. One or more of the essential components may be absent or present in insufficient amounts preventing normal fertility from being expressed. Maw (1954) has described a condition of this nature, regulated by a single autosomal recessive gene, in which insufficient riboflavin was deposited in the egg by the hen resulting in greatly reduced hatchability of fertile eggs. If there is a deficiency in the seminal plasma of RR males, it might be revealed through the use of heterospermic insemination, which involves concurrent insemina-
1552
R. D. CRAWFORD AND J. R. SMYTH, JR.
MATERIALS AND METHODS
The source and management of birds derived from the Ottawa Meat Control strain for use in these studies have been described elsewhere (Crawford and Smyth, 1964a). Two generations of the strain were studied. The comb genotype (RR, Rr, rr) of each individual was known and its was used as the basis of all comparisons.
Exchanging Seminal Plasmas: To determine whether the cause of low fertility of sperm from RR males might lie in some fault of the seminal plasma, native seminal plasma was replaced just prior to insemination by that from males of a different genotype, and the effects studied on the basis of duration of fertilizing ability. The procedure in preparing semen treatments was as follows. Semen was collected from a minimum of six RR males and then
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Diego on June 1, 2015
Homospermic vs. Heterospermic Insemination: An experiment was conducted to determine whether the use of pooled semen from several males (heterospermic insemination) would result in duration of fertility different from that which would be obtained in pedigree matings (homospermic insemination). Semen was collected from ten RR males and was used immediately for pedigree artificial insemination of three White Leghorn females per male. Semen sample remainders, regardless of volume, were pooled and gently mixed. Thirty females were bred using the pooled semen. The entire operation required less than an hour. Semen was then collected from seven rr males and it was treated in a similar manner, the operation requiring about 45 minutes. Eggs were set the day after they were laid, incubated for six days, and candled to determine fertility. Those which appeared to be infertile were broken open for verification.
pooled. Another pooled sample was obtained from a minimum of five rr males. The sample from the rose comb birds was divided into three equal portions. One portion was placed in a covered container and held at room temperature. The remaining two portions and the sample from the single comb birds were centrifuged at approximately 1200 R.C.F. for ten minutes. One portion of the semen from rose comb males was reconstituted by stirring gently with a glass rod. Seminal plasma was removed from the other portion and discarded. Seminal plasma from the semen of single comb males was removed and passed through a Millipore filter (Millipore Filter Corp., Bedford, Mass.) to ensure that all sperm cells were removed, and it was added to the packed rose comb sperm cells, replacing the volume of seminal plasma which had been removed. This sample was then reconstituted by stirring gently with a glass rod. The three treatments thus comprised the following: untreated RR semen (control), RR sperm suspended in RR seminal plasma, and RR sperm suspended in rr seminal plasma. Single comb females of an unrelated stock were inseminated immediately after the treatments had been prepared. The entire procedure, from collection of semen to insemination, required about an hour. The process was then repeated using single comb males as the sperm source. Treatments comprised untreated rr semen (control), rr sperm suspended in rr seminal plasma, and rr sperm suspended in RR seminal plasma. A minimum of seven single comb and six rose comb males contributed to the pooled semen samples. Ten females were bred using semen of each of the six treatments on each of three days, except that on the last day only seven females were available for each of the single comb treatments. Thus, a total of 171 hens was bred, 30 in each of the three rose comb
SEMEN QUALITY AND ROSE COMB
groups and 27 in each of the three involving sperm cells from single comb males. Eggs were incubated the day after laying, fertility was determined by candling after six days of incubation, and eggs appearing infertile were broken open for verification.
under oil immersion at 800X magnification. Two hundred cells were counted per slide from a minimum of three fields. Samples from one single comb male were grossly abnormal and have been omitted from the comparison. Abnormal sperm were classified following the descriptions given by Kamar and Badreldin (1959). Since the proportions of each type of abnormality were very small and did not differ among the groups, they have been combined in the summary. The incidence of crooked-necked sperm, as described by Saeki (1960), was recorded independently of other abnormalities. In studying sperm motility, comparisons were made on all males on three consecutive days. A small drop of semen, mixed with 0.9% NaCl solution, was placed on a glass slide, covered with a cover glass, and examined at 430 X magnification. Motility was rated using a system based on that of Parker et al. (1946). Numerical ratings were assigned as follows: 5—most live sperm showing very vigorous movement. 4—most live sperm showing vigorous movement. 3—most live sperm showing moderate movement. 2—most live sperm showing slow movement. 1—most live sperm showing very slow movement. No samples were encountered which showed a total lack of motility. Sperm concentration was measured by semen optical density, using the technique described by Taneja and Gowe (1961). Three consecutive daily measurements were made for each of the 48 males. The values obtained were much higher than those obtained by Taneja and Gowe (1961) in a study of the same strain. It is possible that the difference arose through the use of
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Diego on June 1, 2015
Evaluating Semen Quality: Sixteen males of each of the three genotypes (RR, Rr, and rr) were evaluated on the basis of semen quality. The studies were conducted over a period of three months. During this interval all of the males were held in individual cages. Semen for volume measurements was collected using the method of Burrows and Quinn (1937). Collections were made on five consecutive days. Each sample, collected in a glass vial, was measured using a 1 ml. syringe calibrated in 0.01 ml. units. For all subsequent studies of semen quality, semen was collected in a manner which would avoid including excessive amounts of "transparent fluid," since this fluid has been reported to have an adverse effect upon fertilizing ability of the spermatozoa (Nishiyama, 1955; Lake, 1956). This was accomplished by collecting a single drop of semen in a glass vial, using a minimum of manual pressure. To determine the proportions of live and dead sperm, stained slides were prepared using the method described by Blom (1950). The preparations were observed under the microscope at 880 X magnification within a few hours. A slide was prepared for each male on three consecutive days. A minimum of three fields was examined on each slide, counting all cells in the field and counting between 200 and 300 cells per slide. The slides prepared for comparisons of live vs. dead sperm were also used to determine the percentage of abnormal sperm. Three slides from each male were examined
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R. D. CRAWFORD AND J. R. SMYTH, JR. TABLE 2.—Analyses of variance of data comparing duration of fertility from pedigreed vs. pooled semen from RR and rr males d.f.
Source
Including all matings producing eggs Genotypes 1 Treatments 1 Interaction 1 Individuals 98 Omitting sterile matings Genotypes Treatments Interaction Individuals
1 1 1 78
F
1,222.72 1.20 10.92 33.52
36.467** 0.036 0.325
481.90 0.46 1.28 20.34
23.692** 0.022 0.062
** Significant at the 1 percent level.
samples were mixed then perhaps those cells from an unfavorable environment would receive enough "rejuvenation" when placed in contact with seminal plasma from normal RR males to improve their fertilizing ability. Data from this experiment are shown in Table 1. It is apparent that fertility of semen from RR males was not improved by pooling the samples, nor was that of the rr control group altered. The analyses shown in Table 2 indicated highly significant (P < .01) differences between genotypes. However, treatment and interaction mean squares did not approach statistical significance.
RESULTS AND DISCUSSION
Homospermtc vs. Heterospermtc Insemination: In the present investigation, concerned with the effects of the seminal fluids on sperm function, duration of fertility from homospermic insemination was compared with that from matings using heterospermic insemination. Since a few of the RR males used here had normal fertility (Crawford and Smyth, 1964a), it was reasoned that at least some of the seminal plasmas would be normal. If the semen
Exchanging Seminal Plasmas: In the published studies of heterospermic insemina-
TABLE 1.—Duration of fertility of pedigreed versus pooled semen from RR and rr males mated by artificial insemination to rr females Pedigreed semen Males, no.
Females, no.
Pooled semen
Duration of fertility days
Males, no.
Females, no.
Duration of fertility days
Including all matings producing eggs RR males 10 30 rr males 7 21
5.20 12.90
10 7
30 21
5.53 11.90
Omitting sterile matings RR males 10 rr males 7
7.80 12.90
10 7
21 20
7.90 12.50
20 21
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cuvettes of different dimensions. Those used in the present study were 19 X 105 mm. It is also possible that the method of collecting semen was not the same in the two studies, differences in the relative amounts of transparent fluid giving rise to the reported differences in concentration. Methylene blue reduction times of semen samples were compared to obtain an estimated measure of metabolic rate of sperm cells. The method used was that of Beck and Salisbury (1943) for bull semen, modified by Bogdonoff and Shaffner (1954) for use with chickens. Methylene blue reduction time was recorded as the time (in 30 second intervals) required for the appearance of a definite yellow color. Samples which did not show any color change after 30 minutes in the water bath were discarded. Duplicate samples from each male were processed on two consecutive days.
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SEMEN QUALITY AND ROSE COMB TABLE 3—Effect of interchanging seminal plasma on duration of fertility of sperm from
RR and rr males, breeding rr females by artificial insemination using pooled semen Including all Males, no.
Treatment
Matings, no.
Duration of fertility, days
, , .. Matings,
24 26 27 25 24 27
2.70 2.61 2.85 8.32 8.50 9.33
11 12 14 22 22 26
rr semen untreated rr sperm & rr plasma rr sperm & RR plasma
tion, no attempt appears to have been made to determine which component of the semen confers the reproductive advantage, assuming that use of this method increases conception rate, as reported by Beatty (1960). An experiment was conducted in the present investigation to determine whether the seminal plasma might be at fault. Pooled samples of semen from males of two comb genotypes were used throughout, seminal fluids from these pooled samples being interchanged. Results of this experiment are shown in Table 3 and the analyses in Table 4. When the sperm were obtained from RR males and the seminal plasma from rr males, there was no improvement in duration of fertility as compared to that from semen treatments involving only homozygous rose comb males. Similarly, no difference was obtained from controls using sperm from rr males mixed with seminal plasma from RR
Duration of f
;i;
days 5.90 5.66 5.50 9.45 9.27 9.69
males. It is concluded that under the conditions of this experiment, there was no indication that substituting seminal plasma with that from normal (rr) males improved the duration of fertility of sperm cells from RR males, implying that the seminal fluid is not involved in the problem of low fertility in homozygous rose comb males. It should be pointed out, however, that in this experiment the semen treatments were prepared and inseminated within an hour of collecting the samples. If the treatments had been incubated or left standing for a period of several hours before insemination, the sperm cells from RR males might have had more time to undergo the hypothesized "rejuvenation" and might have given quite different results. Additional investigations should be conducted before the possibility of the involvement of faulty seminal fluid is completely eliminated.
TABLE 4.—Analyses of variance of data on the effect of interchanging seminal plasma on i duration of fertility of sperm from RR and rr males Including all data
Omitting sterile matings
Source d.f.
m.s.
d.f.
m.s.
F 0.067
Treatments using sperm from RR males Treatments 2 0.375 Individuals 74 11.872
0.031
2 34
0.515 7.620
Treatments using sperm from rr males Treatments 2 7.650 Individuals 73 15.225
0.502
2 67
1.060 9.363
•
0.113
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8RR 8RR f8RR \6 rr 9 rr 9rr )9rr \6RR
RR semen untreated RR sperm & RR plasma RR sperm & rr plasma
Omitting sterile matings
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R. D. CRAWFORD AND J. R. SMYTH, JR. TABLE S.Semen
Semen volume, cc. Dead sperm, percent Abnormal sperm, percent Crooked-necked sperm, percent Sperm motility rating Sperm concentration, optical density Methylene blue reduction time, minutes
quality of RR, Rr, and rr males RR males
Rr males
rr males
0.63 (16)* 41.88 (16) 2.15 (16) 50.76 (16) 3.41 (16) 0.516(16) 9.89 (16)
0.60 (16) 49.46 (16) 2.16 (16) 46.37 (16) 3.74 (16) 0.488 (16) 9.20 (15)
0.64 (16) 41.03 (16) 2.64 (15) 53.00 (15) 3.83 (16) 0.436(16) 11.31 (14)
* Number of males per group.
that of males of the other genotypes. Perhaps if a more refined measurement technique had been used, larger differences would have been found. Two new techniques for quantitative evaluation of sperm motility have been described recently (Gassner et al., 1959; Troll and Goldzieher, 1962). It might be profitable to re-examine sperm motility of males of the three genotypes using these quantitative measures. Sperm concentration from RR males was higher than that from males of the other genotypes although the differences were not statistically significant. A similar phenomenon has been noted by Smyth (1963) in semen quality comparisons of White Plymouth Rocks selected for high and low duration of fertility; in this case, low line
TABLE 6.—A nalyses of variance of semen quality data Source
d.f.
Semen volume, cc.
Genotypes Individuals
2 45
0.0092 0.0331
0.2786
Dead sperm, percent
Genotypes Individuals
2 45
132.9177 116.4299
1.1416
Abnormal sperm, percent
Genotypes Individuals
2 44
4.1000 11.2106
0.3657
Crooked-necked sperm, percent
Genotypes Individuals
2 44
66.9850 162.0506
0.4133
Sperm motility rating
Genotypes Individuals
2 45
0.7772 0.7546
1.0299
Sperm concentration, optical density
Genotypes Individuals
2 45
0.0263 0.0224
1.1723
Methylene blue reduction time, minutes
Genotypes Individuals
2 42
16.7041 11.5560
1.4454
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Evaluating Semen Quality: Since it has been shown that low fertility of RR males is due to some effect of the RR male host on the sperm cells (Crawford and Smyth, 1964b), it might be expected that a study of semen quality would reveal the nature of this effect. Various measures of semen quality have been used to compare males of RR, Rr, and rr comb genotypes. Results of these studies are shown in Table 5 and analyses of the data appear in Table 6. It is evident from inspection of these tables that none of the measures of semen quality revealed any statistically significant differences among the three groups of males. It should be noted that the average motility of RR males was somewhat lower than
SEMEN QUALITY AND ROSE COMB
males consistently demonstrated higher sperm concentration. SUMMARY
REFERENCES Beatty, R. A., 1960. Fertility of mixed semen from different rabbits. J. Reprod. Fert. 1: 52-60. Beck, G. H., and G. W. Salisbury, 1943. Rapid methods of estimating the quality of bull semen. J. Dairy Sci. 26: 483-493. Blom, E., 1950. A one-minute live-dead sperm stain by means of eosin-nigrosin. Fert. Ster. 1: 176-177. Bogdonoff, P. D., Jr., and C. S. Shaffner, 1954. The effect of pH on in vitro survival, metabolic activity, and fertilizing capacity of chicken semen. Poultry Sci. 33 : 665-669. Burrows, W. H., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16: 19-24. Cooper, D. M., and J. G. Rowell, 1958. Relations between fertility, embryonic survival and some semen characteristics in the chicken. Poultry Sci. 37: 699-707. Crawford, R. D., and E. S. Merritt, 1963. The relationship between rose comb and reproduction in the domestic fowl. Can. J. Genet. Cytol. 5: 89-95.
Crawford, R. D., and J. R. Smyth, Jr., 1964a. Studies of the relationship between fertility and the gene for rose comb in the domestic fowl. 1. The relationship between comb genotype and fertility. Poultry Sci. 4 3 : 1009-1017. Crawford, R. D., and J. R. Smyth, Jr., 1964b. Infertility and action of the gene for rose comb in the domestic fowl. Can. J. Genet. Cytol. (in press). Gassner, F. X., J. W. Goldzieher, J. F. Masken and J. L. Hopwood, 1959. The objective measurement of sperm motility. Fert. Ster. 10: 488501. Hess, E. A., T. M. Ludwick, H. E. Rickard and F. Ely, 1958. Some of the effects of heterospermic processing on semen quality and bovine fertility. Fert. Ster. 9: 238-242. Kamar, G. A. R., 1960. The influence of semen characteristics on hatching results of chicken eggs. Poultry Sci. 39: 188-192. Kamar, G. A. R., and A. L. Badreldin, 1959. Sperm morphology and viability. Acta Anat. 39 : 81-83. Lake, P. E., 1956. A retarding factor in the problem of fowl semen storage. Proc. 3rd Intern. Cong. Anim. Prod., Cambridge, Sect. 3 : 104106. Lorenz, F. W., 1959. Reproduction in the domestic fowl: physiology of the male. Chapter 11 in: Reproduction in Domestic Animals, vol. 2. Cole, H. H., and P. T. Cupps, eds. Academic Press, New York, N.Y. Maw, A. J. G., 1954. Inherited riboflavin deficiency in chicken eggs. Poultry Sci. 33 : 216-217. McDaniel, G. R., and J. V. Craig, 1959. Behavior traits, semen measurements and fertility of White Leghorn males. Poultry Sci. 38: 10051014. Nishiyama, H., 1955. Studies on the accessory reproductive organs in the cock. J. Fac. Agr., Kyushu Univ. 10: 277-305. Parker, J. E., F. F. McKenzie and H. L. Kempster, 1946. Fertility in the male domestic fowl. Missouri Agr. Exp. Sta. Res. Bull. 347: 1-50. Saeki, Y., 1960. Crooked-necked spermatozoa in relation to low fertility in the artificial insemination of fowl. Poultry Sci. 39: 1354-1361. Smyth, J. R., Jr., 1963. Unpublished data. Taneja, G. C , and R. S. Gowe, 1961. Spermatozoa concentration in the semen of two breeds of fowl estimated by three different methods. Poultry Sci. 40: 608-615. Troll, J., and J. W. Goldzieher, 1962. Quantitative evaluation of the motility of spermatozoa. Fert. Ster. 13: 72-83.
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In studying the problem of low fertility associated with homozygosity for the gene for rose comb in male domestic fowl, experiments were conducted to compare qualities of the semen and seminal plasma of males of RR, Rr, and rr genotypes. Experiments involving pooled semen and experiments in which seminal plasma from the low fertility group was replaced by that from single comb males indicated that there was no apparent deficiency in seminal fluids of RR males causing their fertility to be depressed. Semen quality of males of the three comb genotypes was compared using semen volume, percent dead sperm, percent abnormal sperm, sperm motility, semen optical density, and methylene blue reduction time as measures of quality. In none of these comparisons were RR males found to differ significantly from Rr or rr males.
1557
T
1
Contribution from Massachusetts Agricultural Experiment Station. 2 Present address: Department of Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan.
tion of a female with semen of two or more males. Literature describing the results obtained by this method has been reviewed by Beatty (1960) and by Hess et al. (1958). Russian proponents of the technique claim that its use results in a variety of beneficial results such as improved motility and viability of spermatozoa, higher conception rate and birth rate, lower mortality of offspring, and more rapid growth. However, these claims have not received general support among Western workers. Literature concerning the relationship between fertility and semen quality in the domestic fowl has been reviewed by Lorenz (1959) and by Kamar (1960). These reviews indicate that the percentage of abnormal sperm, initial motility, metabolic activity, and the percentage of live vs. dead sperm are closely related to fertilizing ability of the semen. However, these measures are usually found to be useful mainly for predicting the fertilizing capacity of individual males; when pooled semen is used, the relationships are not so marked. Two reports (Cooper and Rowell, 1958; McDaniel and Craig, 1959) have indicated that there is no relationship between semen volume and fertility, but Kamar (1960) found a significant positive correlation between these traits using a highly selected group of birds. In the review by Lorenz (1959), it is stated that sperm density has not been shown to account for significant amounts of variation in fertility. Since constant volumes are usually inseminated, a relationship would not normally be expected between sperm concentration and fertility.
1551
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Diego on June 1, 2015
HE low fertility characteristic of White Wyandottes is associated with rose comb and is expressed only by males homozygous (RR) for that comb type (Crawford and Merritt, 1963; Crawford and Smyth, 1964a). Presence of the gene for rose comb in the sperm cell does not decrease fertility of that cell; instead, the low fertility is caused by some deleterious effect of the RR male host body acting on the spermatozoa (Crawford and Smyth, 1964b). To determine whether this effect might be identified in the semen, several experiments have been conducted to compare qualities of semen and seminal plasma of males of RR, Rr, and rr comb genotypes. A possible reason for the low fertility of RR males is that some inadequacy might exist in their seminal fluids. One or more of the essential components may be absent or present in insufficient amounts preventing normal fertility from being expressed. Maw (1954) has described a condition of this nature, regulated by a single autosomal recessive gene, in which insufficient riboflavin was deposited in the egg by the hen resulting in greatly reduced hatchability of fertile eggs. If there is a deficiency in the seminal plasma of RR males, it might be revealed through the use of heterospermic insemination, which involves concurrent insemina-
1552
R. D. CRAWFORD AND J. R. SMYTH, JR.
MATERIALS AND METHODS
The source and management of birds derived from the Ottawa Meat Control strain for use in these studies have been described elsewhere (Crawford and Smyth, 1964a). Two generations of the strain were studied. The comb genotype (RR, Rr, rr) of each individual was known and its was used as the basis of all comparisons.
Exchanging Seminal Plasmas: To determine whether the cause of low fertility of sperm from RR males might lie in some fault of the seminal plasma, native seminal plasma was replaced just prior to insemination by that from males of a different genotype, and the effects studied on the basis of duration of fertilizing ability. The procedure in preparing semen treatments was as follows. Semen was collected from a minimum of six RR males and then
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Diego on June 1, 2015
Homospermic vs. Heterospermic Insemination: An experiment was conducted to determine whether the use of pooled semen from several males (heterospermic insemination) would result in duration of fertility different from that which would be obtained in pedigree matings (homospermic insemination). Semen was collected from ten RR males and was used immediately for pedigree artificial insemination of three White Leghorn females per male. Semen sample remainders, regardless of volume, were pooled and gently mixed. Thirty females were bred using the pooled semen. The entire operation required less than an hour. Semen was then collected from seven rr males and it was treated in a similar manner, the operation requiring about 45 minutes. Eggs were set the day after they were laid, incubated for six days, and candled to determine fertility. Those which appeared to be infertile were broken open for verification.
pooled. Another pooled sample was obtained from a minimum of five rr males. The sample from the rose comb birds was divided into three equal portions. One portion was placed in a covered container and held at room temperature. The remaining two portions and the sample from the single comb birds were centrifuged at approximately 1200 R.C.F. for ten minutes. One portion of the semen from rose comb males was reconstituted by stirring gently with a glass rod. Seminal plasma was removed from the other portion and discarded. Seminal plasma from the semen of single comb males was removed and passed through a Millipore filter (Millipore Filter Corp., Bedford, Mass.) to ensure that all sperm cells were removed, and it was added to the packed rose comb sperm cells, replacing the volume of seminal plasma which had been removed. This sample was then reconstituted by stirring gently with a glass rod. The three treatments thus comprised the following: untreated RR semen (control), RR sperm suspended in RR seminal plasma, and RR sperm suspended in rr seminal plasma. Single comb females of an unrelated stock were inseminated immediately after the treatments had been prepared. The entire procedure, from collection of semen to insemination, required about an hour. The process was then repeated using single comb males as the sperm source. Treatments comprised untreated rr semen (control), rr sperm suspended in rr seminal plasma, and rr sperm suspended in RR seminal plasma. A minimum of seven single comb and six rose comb males contributed to the pooled semen samples. Ten females were bred using semen of each of the six treatments on each of three days, except that on the last day only seven females were available for each of the single comb treatments. Thus, a total of 171 hens was bred, 30 in each of the three rose comb
SEMEN QUALITY AND ROSE COMB
groups and 27 in each of the three involving sperm cells from single comb males. Eggs were incubated the day after laying, fertility was determined by candling after six days of incubation, and eggs appearing infertile were broken open for verification.
under oil immersion at 800X magnification. Two hundred cells were counted per slide from a minimum of three fields. Samples from one single comb male were grossly abnormal and have been omitted from the comparison. Abnormal sperm were classified following the descriptions given by Kamar and Badreldin (1959). Since the proportions of each type of abnormality were very small and did not differ among the groups, they have been combined in the summary. The incidence of crooked-necked sperm, as described by Saeki (1960), was recorded independently of other abnormalities. In studying sperm motility, comparisons were made on all males on three consecutive days. A small drop of semen, mixed with 0.9% NaCl solution, was placed on a glass slide, covered with a cover glass, and examined at 430 X magnification. Motility was rated using a system based on that of Parker et al. (1946). Numerical ratings were assigned as follows: 5—most live sperm showing very vigorous movement. 4—most live sperm showing vigorous movement. 3—most live sperm showing moderate movement. 2—most live sperm showing slow movement. 1—most live sperm showing very slow movement. No samples were encountered which showed a total lack of motility. Sperm concentration was measured by semen optical density, using the technique described by Taneja and Gowe (1961). Three consecutive daily measurements were made for each of the 48 males. The values obtained were much higher than those obtained by Taneja and Gowe (1961) in a study of the same strain. It is possible that the difference arose through the use of
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Diego on June 1, 2015
Evaluating Semen Quality: Sixteen males of each of the three genotypes (RR, Rr, and rr) were evaluated on the basis of semen quality. The studies were conducted over a period of three months. During this interval all of the males were held in individual cages. Semen for volume measurements was collected using the method of Burrows and Quinn (1937). Collections were made on five consecutive days. Each sample, collected in a glass vial, was measured using a 1 ml. syringe calibrated in 0.01 ml. units. For all subsequent studies of semen quality, semen was collected in a manner which would avoid including excessive amounts of "transparent fluid," since this fluid has been reported to have an adverse effect upon fertilizing ability of the spermatozoa (Nishiyama, 1955; Lake, 1956). This was accomplished by collecting a single drop of semen in a glass vial, using a minimum of manual pressure. To determine the proportions of live and dead sperm, stained slides were prepared using the method described by Blom (1950). The preparations were observed under the microscope at 880 X magnification within a few hours. A slide was prepared for each male on three consecutive days. A minimum of three fields was examined on each slide, counting all cells in the field and counting between 200 and 300 cells per slide. The slides prepared for comparisons of live vs. dead sperm were also used to determine the percentage of abnormal sperm. Three slides from each male were examined
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R. D. CRAWFORD AND J. R. SMYTH, JR. TABLE 2.—Analyses of variance of data comparing duration of fertility from pedigreed vs. pooled semen from RR and rr males d.f.
Source
Including all matings producing eggs Genotypes 1 Treatments 1 Interaction 1 Individuals 98 Omitting sterile matings Genotypes Treatments Interaction Individuals
1 1 1 78
F
1,222.72 1.20 10.92 33.52
36.467** 0.036 0.325
481.90 0.46 1.28 20.34
23.692** 0.022 0.062
** Significant at the 1 percent level.
samples were mixed then perhaps those cells from an unfavorable environment would receive enough "rejuvenation" when placed in contact with seminal plasma from normal RR males to improve their fertilizing ability. Data from this experiment are shown in Table 1. It is apparent that fertility of semen from RR males was not improved by pooling the samples, nor was that of the rr control group altered. The analyses shown in Table 2 indicated highly significant (P < .01) differences between genotypes. However, treatment and interaction mean squares did not approach statistical significance.
RESULTS AND DISCUSSION
Homospermtc vs. Heterospermtc Insemination: In the present investigation, concerned with the effects of the seminal fluids on sperm function, duration of fertility from homospermic insemination was compared with that from matings using heterospermic insemination. Since a few of the RR males used here had normal fertility (Crawford and Smyth, 1964a), it was reasoned that at least some of the seminal plasmas would be normal. If the semen
Exchanging Seminal Plasmas: In the published studies of heterospermic insemina-
TABLE 1.—Duration of fertility of pedigreed versus pooled semen from RR and rr males mated by artificial insemination to rr females Pedigreed semen Males, no.
Females, no.
Pooled semen
Duration of fertility days
Males, no.
Females, no.
Duration of fertility days
Including all matings producing eggs RR males 10 30 rr males 7 21
5.20 12.90
10 7
30 21
5.53 11.90
Omitting sterile matings RR males 10 rr males 7
7.80 12.90
10 7
21 20
7.90 12.50
20 21
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cuvettes of different dimensions. Those used in the present study were 19 X 105 mm. It is also possible that the method of collecting semen was not the same in the two studies, differences in the relative amounts of transparent fluid giving rise to the reported differences in concentration. Methylene blue reduction times of semen samples were compared to obtain an estimated measure of metabolic rate of sperm cells. The method used was that of Beck and Salisbury (1943) for bull semen, modified by Bogdonoff and Shaffner (1954) for use with chickens. Methylene blue reduction time was recorded as the time (in 30 second intervals) required for the appearance of a definite yellow color. Samples which did not show any color change after 30 minutes in the water bath were discarded. Duplicate samples from each male were processed on two consecutive days.
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SEMEN QUALITY AND ROSE COMB TABLE 3—Effect of interchanging seminal plasma on duration of fertility of sperm from
RR and rr males, breeding rr females by artificial insemination using pooled semen Including all Males, no.
Treatment
Matings, no.
Duration of fertility, days
, , .. Matings,
24 26 27 25 24 27
2.70 2.61 2.85 8.32 8.50 9.33
11 12 14 22 22 26
rr semen untreated rr sperm & rr plasma rr sperm & RR plasma
tion, no attempt appears to have been made to determine which component of the semen confers the reproductive advantage, assuming that use of this method increases conception rate, as reported by Beatty (1960). An experiment was conducted in the present investigation to determine whether the seminal plasma might be at fault. Pooled samples of semen from males of two comb genotypes were used throughout, seminal fluids from these pooled samples being interchanged. Results of this experiment are shown in Table 3 and the analyses in Table 4. When the sperm were obtained from RR males and the seminal plasma from rr males, there was no improvement in duration of fertility as compared to that from semen treatments involving only homozygous rose comb males. Similarly, no difference was obtained from controls using sperm from rr males mixed with seminal plasma from RR
Duration of f
;i;
days 5.90 5.66 5.50 9.45 9.27 9.69
males. It is concluded that under the conditions of this experiment, there was no indication that substituting seminal plasma with that from normal (rr) males improved the duration of fertility of sperm cells from RR males, implying that the seminal fluid is not involved in the problem of low fertility in homozygous rose comb males. It should be pointed out, however, that in this experiment the semen treatments were prepared and inseminated within an hour of collecting the samples. If the treatments had been incubated or left standing for a period of several hours before insemination, the sperm cells from RR males might have had more time to undergo the hypothesized "rejuvenation" and might have given quite different results. Additional investigations should be conducted before the possibility of the involvement of faulty seminal fluid is completely eliminated.
TABLE 4.—Analyses of variance of data on the effect of interchanging seminal plasma on i duration of fertility of sperm from RR and rr males Including all data
Omitting sterile matings
Source d.f.
m.s.
d.f.
m.s.
F 0.067
Treatments using sperm from RR males Treatments 2 0.375 Individuals 74 11.872
0.031
2 34
0.515 7.620
Treatments using sperm from rr males Treatments 2 7.650 Individuals 73 15.225
0.502
2 67
1.060 9.363
•
0.113
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8RR 8RR f8RR \6 rr 9 rr 9rr )9rr \6RR
RR semen untreated RR sperm & RR plasma RR sperm & rr plasma
Omitting sterile matings
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R. D. CRAWFORD AND J. R. SMYTH, JR. TABLE S.Semen
Semen volume, cc. Dead sperm, percent Abnormal sperm, percent Crooked-necked sperm, percent Sperm motility rating Sperm concentration, optical density Methylene blue reduction time, minutes
quality of RR, Rr, and rr males RR males
Rr males
rr males
0.63 (16)* 41.88 (16) 2.15 (16) 50.76 (16) 3.41 (16) 0.516(16) 9.89 (16)
0.60 (16) 49.46 (16) 2.16 (16) 46.37 (16) 3.74 (16) 0.488 (16) 9.20 (15)
0.64 (16) 41.03 (16) 2.64 (15) 53.00 (15) 3.83 (16) 0.436(16) 11.31 (14)
* Number of males per group.
that of males of the other genotypes. Perhaps if a more refined measurement technique had been used, larger differences would have been found. Two new techniques for quantitative evaluation of sperm motility have been described recently (Gassner et al., 1959; Troll and Goldzieher, 1962). It might be profitable to re-examine sperm motility of males of the three genotypes using these quantitative measures. Sperm concentration from RR males was higher than that from males of the other genotypes although the differences were not statistically significant. A similar phenomenon has been noted by Smyth (1963) in semen quality comparisons of White Plymouth Rocks selected for high and low duration of fertility; in this case, low line
TABLE 6.—A nalyses of variance of semen quality data Source
d.f.
Semen volume, cc.
Genotypes Individuals
2 45
0.0092 0.0331
0.2786
Dead sperm, percent
Genotypes Individuals
2 45
132.9177 116.4299
1.1416
Abnormal sperm, percent
Genotypes Individuals
2 44
4.1000 11.2106
0.3657
Crooked-necked sperm, percent
Genotypes Individuals
2 44
66.9850 162.0506
0.4133
Sperm motility rating
Genotypes Individuals
2 45
0.7772 0.7546
1.0299
Sperm concentration, optical density
Genotypes Individuals
2 45
0.0263 0.0224
1.1723
Methylene blue reduction time, minutes
Genotypes Individuals
2 42
16.7041 11.5560
1.4454
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Evaluating Semen Quality: Since it has been shown that low fertility of RR males is due to some effect of the RR male host on the sperm cells (Crawford and Smyth, 1964b), it might be expected that a study of semen quality would reveal the nature of this effect. Various measures of semen quality have been used to compare males of RR, Rr, and rr comb genotypes. Results of these studies are shown in Table 5 and analyses of the data appear in Table 6. It is evident from inspection of these tables that none of the measures of semen quality revealed any statistically significant differences among the three groups of males. It should be noted that the average motility of RR males was somewhat lower than
SEMEN QUALITY AND ROSE COMB
males consistently demonstrated higher sperm concentration. SUMMARY
REFERENCES Beatty, R. A., 1960. Fertility of mixed semen from different rabbits. J. Reprod. Fert. 1: 52-60. Beck, G. H., and G. W. Salisbury, 1943. Rapid methods of estimating the quality of bull semen. J. Dairy Sci. 26: 483-493. Blom, E., 1950. A one-minute live-dead sperm stain by means of eosin-nigrosin. Fert. Ster. 1: 176-177. Bogdonoff, P. D., Jr., and C. S. Shaffner, 1954. The effect of pH on in vitro survival, metabolic activity, and fertilizing capacity of chicken semen. Poultry Sci. 33 : 665-669. Burrows, W. H., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16: 19-24. Cooper, D. M., and J. G. Rowell, 1958. Relations between fertility, embryonic survival and some semen characteristics in the chicken. Poultry Sci. 37: 699-707. Crawford, R. D., and E. S. Merritt, 1963. The relationship between rose comb and reproduction in the domestic fowl. Can. J. Genet. Cytol. 5: 89-95.
Crawford, R. D., and J. R. Smyth, Jr., 1964a. Studies of the relationship between fertility and the gene for rose comb in the domestic fowl. 1. The relationship between comb genotype and fertility. Poultry Sci. 4 3 : 1009-1017. Crawford, R. D., and J. R. Smyth, Jr., 1964b. Infertility and action of the gene for rose comb in the domestic fowl. Can. J. Genet. Cytol. (in press). Gassner, F. X., J. W. Goldzieher, J. F. Masken and J. L. Hopwood, 1959. The objective measurement of sperm motility. Fert. Ster. 10: 488501. Hess, E. A., T. M. Ludwick, H. E. Rickard and F. Ely, 1958. Some of the effects of heterospermic processing on semen quality and bovine fertility. Fert. Ster. 9: 238-242. Kamar, G. A. R., 1960. The influence of semen characteristics on hatching results of chicken eggs. Poultry Sci. 39: 188-192. Kamar, G. A. R., and A. L. Badreldin, 1959. Sperm morphology and viability. Acta Anat. 39 : 81-83. Lake, P. E., 1956. A retarding factor in the problem of fowl semen storage. Proc. 3rd Intern. Cong. Anim. Prod., Cambridge, Sect. 3 : 104106. Lorenz, F. W., 1959. Reproduction in the domestic fowl: physiology of the male. Chapter 11 in: Reproduction in Domestic Animals, vol. 2. Cole, H. H., and P. T. Cupps, eds. Academic Press, New York, N.Y. Maw, A. J. G., 1954. Inherited riboflavin deficiency in chicken eggs. Poultry Sci. 33 : 216-217. McDaniel, G. R., and J. V. Craig, 1959. Behavior traits, semen measurements and fertility of White Leghorn males. Poultry Sci. 38: 10051014. Nishiyama, H., 1955. Studies on the accessory reproductive organs in the cock. J. Fac. Agr., Kyushu Univ. 10: 277-305. Parker, J. E., F. F. McKenzie and H. L. Kempster, 1946. Fertility in the male domestic fowl. Missouri Agr. Exp. Sta. Res. Bull. 347: 1-50. Saeki, Y., 1960. Crooked-necked spermatozoa in relation to low fertility in the artificial insemination of fowl. Poultry Sci. 39: 1354-1361. Smyth, J. R., Jr., 1963. Unpublished data. Taneja, G. C , and R. S. Gowe, 1961. Spermatozoa concentration in the semen of two breeds of fowl estimated by three different methods. Poultry Sci. 40: 608-615. Troll, J., and J. W. Goldzieher, 1962. Quantitative evaluation of the motility of spermatozoa. Fert. Ster. 13: 72-83.
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In studying the problem of low fertility associated with homozygosity for the gene for rose comb in male domestic fowl, experiments were conducted to compare qualities of the semen and seminal plasma of males of RR, Rr, and rr genotypes. Experiments involving pooled semen and experiments in which seminal plasma from the low fertility group was replaced by that from single comb males indicated that there was no apparent deficiency in seminal fluids of RR males causing their fertility to be depressed. Semen quality of males of the three comb genotypes was compared using semen volume, percent dead sperm, percent abnormal sperm, sperm motility, semen optical density, and methylene blue reduction time as measures of quality. In none of these comparisons were RR males found to differ significantly from Rr or rr males.
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