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Diluents for Turkey Semen
Diluents for Turkey Semen A. VAN TlENHOVEN,1 R. G. D . STEEL2 AND S. A. DUCHAINE1 Agricultural Experiment Station at Cornell University, Ithaca, New York (Received for publication June 27, 1957)
HERE are, to our knowledge, no reports in published literature on the effect of antibiotics and glycine on turkey spermatozoa. The reports on the effect of these additions to avian semen have been concerned with rooster semen (Smith, 1949; Lorenz and Tyler, 1951; Terbush, 1952; Qureshi, 1952). Van Tienhoven and Steel (1957) reported on experiments with turkey semen diluted with three different diluents at dilution rates ranging from 1:1 to 1:15 in which no differences among dilution rates were detected. However, it has been possible to obtain fertility at higher dilution rates with turkey than with rooster semen (van Tienhoven, unpublished), thus indicating differences between semen of these species.
In the fertility experiment 117 hens were distributed at random over 13 pens of 9 hens each. Within each pen each treatment was represented once and the hens were assigned at random to the treatments. The random assignments were used because no prediction could be made of a particular hen's expected fertility. Inseminations in pens 2-12 were made on February 4, 6 and 25 while in pens 13 and 14, which came into production later, the dates of insemination were February 14 and 25. Three hundredths of a c.c. of either diluted or undiluted semen was inseminated. We determined fertility according to the methods outlined previously (van Tienhoven and Steel, 1957). The maximum length of storage of eggs was 10 days.
We decided to study the effect of added glycine and antibiotics on both fertilizing capacity and livability of turkey semen in different diluents using dilution rates of 1:15.
Sperm concentration determinations were made according to the method of Salisbury el al. (1943). The optical density of semen diluted 1:500 was determined at a wavelength of 600 ran. In our laboratory the calibration curve for this procedure was made with the aid of hemocytometer counts according to Phillips and Wiegers (1952). The regression found can be expressed by the equation:
MATERIALS AND METHODS In all experiments semen from 15-20 toms was pooled and the semen diluted 1:15 with each of the diluents in Table 1. Dilutions were completed within 1 hour after the start of collection and the inseminations were completed within 70 minutes after dilution. Motility examinations and other laboratory procedures necessary for the determinations were finished within 30 minutes after dilution. 1
2
Department of Poultry Husbandry.
Department of Plant Breeding, Biometrics Unit.
F=1984.6X-141.2; r = 0.865 In which F = sperm concentration X 10,000/mm3 X = optical density at 600 rafi r = correlation coefficient The regression coefficient was highly significant (P<0.01). The work of Kosin and Wheeler (1956) shows that estimates made according to the method of Phillips
47
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T
48
A. VAN TIENHOVEN, R. G. D. STEEL AND S. A. DUCHAINE TABLE 1.—Composition of diluents used in experiments 1 and 2 Constituents
No.
Tyrode,* Milkf parts
Tyrode (T) Tyrode+Glycine (TG) Tyrode+Antibiotics (TA) Tyrode+Glycine+Antibiotics (TGA) Milk (M) Milk+Glycine (MG) Milk+Antibiotics (MA) Milk+Glycine+Antibiotics (MGA)
Clvcine i P e n ' c il" Strep to- SulfanilIA7 ™ lin> mycin, amide, units cart?' / mcS-/ m§-/ ^ ml. ml. ml.
LUU
31 100 31
— — — —
.
19
— —.
100 31 100 31
— 19
1,000 1,000
20 20
3 3
—
—. —.
— —.
— —
19
— 19
1,000 1,000
20 20
3 3
* Tyrode according to Bogdonoff and Shaffner (1954) p H = 6 . 8 . t Milk according to Almquist et al. (1954) p H = 6 . 7 . t Glycine 0.167 m. is isotonic with the Tyrode solution used.
and Wiegers (1952) may be too low. This publication came to our attention after the work on this experiment was completed. Methylene blue reduction time (MBRT) and motility were determined according to methods described previously (van Tienhoven and Steel, 1957). For the dead sperm count a total of 400 sperm were counted on each of duplicate slides, while in the motility determinations an incubation stage was used. For the dead sperm count 1 drop of the diluted semen was mixed with about 10 drops of a nigrosin-eosin stain, consisting of 5% nigrosin and 1% eosin dissolved in Tyrode solution. The sperm were exposed to the stain for two minutes and the smear made by pulling two slides apart. The slides were immediately dried by passing them through a flame and by gentle blowing. This method is an adaptation of the methods used for bull sperm by Swanson and Bearden (1951) and by Bade et al. (1956) for turkey sperm. A total of 400 cells was counted on each slide and precautions were taken that the results obtained on one slide were unknown to the observer while the replicate slide was
counted. Campbell et al. (1956) have shown that knowledge of the count on one slide may introduce bias when the second count is made. All counts were made by one person (T.N.). 1 EXPERIMENTS AND RESULTS
Experiment 1: We studied the fertilizing capacity of the semen after dilution with each of the diluents given in Table 1. The concentrations of streptomycin and penicillin are the same as used by Terbush (1952). The sulfanilamide concentration is the same as used for bull semen (Knodt and Salisbury, 1946). The number of sperm inseminated varied on the different insemination dates between 10.34-14.79 million sperm per insemination. The results of the experiment are presented in Table 2. Although 13 hens were inseminated on each treatment, some failed to lay in the trapnest. This explains the variation in the number of hens inseminated in each treatment group. Because of the rather high incidence of hens laying infertile eggs in some treatments, we decided to test whether the 1 Thanks are due to Mr. T. Noszlopi for his help in these experiments.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
1 2 3 4 5 6 7 8
Description
49
DILUENTS FOR TURKEY SEMEN TABLE 2.—The effect of diluents upon fertilizing ca-
pacity of turkey semen Eggs Diluent
Fertility Percent
257 277 223 234 261 208 267 264 230
57.6 67.9 77.1 50.4 52.9 21.6 28.8 15.5 89.7
T TG TA TGA M MG MA MGA Undil.
— Hens inseminated 12 13 13 11 13 13 13 13 11
infertility was due to the treatment or to inherent infertility of the hens. All these hens were subsequently inseminated with undiluted semen and those from which eggs were obtained proved to be fertile. We therefore included all hens in the analysis of the data. The missing data, due to failure to lay in the nest, broodiness, etc., made an analysis of variance of the percentage fertile eggs cumbersome. We therefore decided to analyze the data by a series of chi square tests (Snedecor, 1956) rather than by an analysis of variance. Some biological interactions are so clearcut that no tests of significance were made.
The fertility trial shows clearly that: 1) The differences between the milk and Tyrode diluents are small when no glycine or antibiotics are added. Although the fertility levels obtained were lower than in our previ-
TABLE 3.—The effect of various diluents on semen livability and metabolic activity Day 2
Day 1 Diluent
T TXG TXA
TXGXA M MXG MXA
MXGXA Tvs. M G
(T-M)G (T-M) A GXA (T-M) GXA TXGXA MXGXA Samples
MBRT, nun.
Dead/ - 400 Rate counted
Motility
%
MBRT, mm.
Day 3
Motility
%
Dead/ • 400 Rate counted
Motility
MBRT, mm.
%
Rate 0
4.00 3.75 3.21 3.71
65.0 56.7 70.0 51.7
3.40 3.25 3.25 3.09
80.9 80.4 95.3 75.4
33.25 16.29 15.25 32.89
10.8 16.7 16.7
1.10 1.16 1.33 1.00
102.8 134.5 146.3 142.6
55.5 47.4 52.0 54.3
0 1 1.4 0
4.41 5.13 5.80 5.77
56.7 55.0 53.3 51.7
3.33 3.40 3.00 3.25
39.3 36.1 39.8 47.7
11.32 11.19 16.63 10.56
9.2
1.00 1.83 1.16 1.92
88.7 141.1 117.4 137.3
22.2 20.5 40.7 32.55
1 1 0 5
0
NS NS NS NS NS NS NS
<0.01
<0.01
NS NS NS NS NS NS
NS NS NS NS NS NS NS
NS NS NS NS NS NS
NS NS NS NS
NS NS NS NS NS NS NS
<0.01
<0.05
<0.01
NS
<0.01
<0.01
<0.05 <0.01 <0.01 <0.05 <0.01
8.3
11.7 15.0 20.0
<0.01
NS NS NS NS
NS NS NS NS NS NS NS
<0.05 NS NS NS
NS NS NS NS NS NS NS
<0.01
NS
<0.01
<0.01
NS NS NS NS NS NS NS
<0.05 <0.05 <0.05
<0.01
NS NS
.2 .5
— .3 .3 .8
-
Dead/ 400 counted 210.3 108.5 179.5 133.9 157.0 224.4 231.0 153.0 NS
<0.05 NS NS NS NS NS
NS
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Set
The results posed the question whether it would be feasible to predict the performance of sperm in certain diluents, with respect to fertilizing capacity, from the ability of the diluent to support sperm activity and livability in vitro. The advantages of such a preliminary screening of diluents by in vitro studies are selfevident. The large differences between diluents in this experiment suggested the use of the same diluents for in vitro studies. Experiment 2: After the semen was diluted 1:15 with the various diluents, MBRT, motility and incidence of dead sperm were determined. The semen was subsequently stored at 5°C. and the same determinations made 24 and 48 hours later. The results are given in Table 3. Analyses of variance for the various characteristics studied were computed separately for Day 1, 2, and 3 partly because of heterogeneity of variance. DISCUSSION
50
A. VAN TIENHOVEN, R. G. D. STEEL AND S. A. DUCHAINE
4) Although the T.A. diluent gave better results than the other diluents, the fertility level obtained with this diluent is lower than that obtained with undiluted semen. 5) In the Tyrode solution there was no decline in fertility with increasing
hatch number, but with milk diluents the fertility declined in spite of inseminations repeated three weeks after the first insemination. This strongly suggests that the milk diluent may create a condition in the oviduct unfavorable to fertilization by other sperm also diluted with milk. We have not investigated whether this would also hold true if undiluted semen were used for the later inseminations. 6) The differences between hatches were highly significant, probably due mainly to the effect discussed under 5. 7) The differences between pens are highly significant even when account is taken of the fact that in some pens more birds failed to lay than in others. The data are given in Table 4. We cannot discover any physiological reason why there is a pen effect on fertility. In our previous work we showed that the time between collection and insemination had little or no effect on subsequent fertility. In the present experiments care was taken that the order in TABLE 4.—The effect of pen on fertilizing capacity of turkey semen Eggs
2 3 4 5 6 7 8 9 10 11 12 13 14
Set
Fertile
no. 133 202 230 215 124 209 171 162 166 216 187 105 101
61.7 60.0 36.1 48.4 62.1 55.0 62.0 53.7 29.5 51.4 47.6 70.5 52.5
%
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ous experiment (van Tienhoven and Steel, 1957), the comparison did not lead to different conclusions. 2) The addition of antibiotics to Tyrode diluent caused increased fertility but when added to milk it caused a decrease. This interaction of diluents and antibiotics i.e. the failure of the addition of antibiotics to the two diluents to produce the same change in fertility, was highly significant (<0.01). Similarly, glycine addition to the Tyrode diluent resulted in better fertility than obtained with Tyrode alone, but when glycine was added to milk it had a deleterious effect on fertilizing capacity. This diluent glycine interaction was highly significant ( P < 0.01). 3) The combination of glycine and antibiotics added to Tyrode solution resulted in a fertility not significantly different from the fertility obtained with semen diluted with Tyrode solution. The combination of glycine and antibiotics added to milk resulted in a further depression of fertility, below that found with the milk-glycine or milk-antibiotics diluents. The interaction of diluents and antibiotics plus glycine was highly significant (P<0.01). The three-way interaction implied by this paragraph as related to discussion item 2 was not tested for significance because the magnitudes of the differences involved made such a calculation superfluous.
DILUENTS FOR TURKEY SEMEN
which the pens were inseminated was rotated.
1) On Day 1 the semen diluted with milk diluents had a longer MBRT than semen diluted with Tyrode diluents. This difference is small and may well be due to the fact that the end point in the milk diluent is not as sharp as in the Tyrode solution. 2) The incidence of dead sperm in milk diluents on Day 1 is smaller than in the Tyrode diluents. This difference was not due to interference of the milk with the staining procedure as such. This was demonstrated for semen diluted with Tyrode; just before the stain was added, one or two drops of milk were added and the semen processed as usual. The same was done for semen diluted with milk and subsequent addition of Tyrode just before staining. The results obtained with the Tyrode and milk diluent seem to indicate that the dilution with milk poses less of a "shock" to the sperm than the dilution with Tyrode diluents. However, on further storage there is no significant difference between the diluents.
3) The MBRT is considerably shorter in milk than in Tyrode diluents on Day 2. Although we have not made any bacterial counts in this study, it seems unlikely that the difference between the two diluents is due to greater bacterial growth in the milk diluent. If such were the case antibiotics which suppress bacterial growth (Smith, 1949) might be expected to affect MBRT because the latter is affected by the presence of bacteria (Frobisher, 1953). As antibiotics did not affect MBRT we conclude, tentatively, that milk diluents support metabolic activity better than Tyrode solution, without affecting livability, as can be seen from the incidence of dead sperm in the two groups of diluents. The addition of glycine, which has been reported to be beneficial in supporting the livability of cock semen at room temperature (Lorenz and Tyler, 1951) and of bull semen at 5°C. (Flipse and Almquist, 1956) has a beneficial effect on fertility when added to Tyrode solution but an inhibitory effect when added to milk. Flipse and Almquist (1956) showed that small amounts of glycine actually are harmful in supporting livability. The concentrations which were beneficial in the trials with bull semen (Flipse and Almquist, 1956) are hypertonic. For fowl semen, at least, the work by Lake (1954) indicates that isotonicity of the diluent may be particularly important. For this reason the concentrations used by us are considerably lower than those used by Flipse and Almquist (1956) but the concentrations are the same as those found to be most beneficial for fowl semen by Lorenz and Tyler (1951). A further study of this phenomenon of a beneficial effect of a hypertonic solution
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A comparison between the results of Experiments 1 and 2 shows that with these diluents, at least, the screening of diluents by in vitro studies was not successful. It is, of course, possible that a diluent which supports livability exceptionally well could be screened out this way and then used for fertility trials. But relatively minor differences or even the rather large differences as found on Day 2 in MBRT (see Table 3) do not give any lead to the future success of a diluent in a fertility trial. The in vitro study (see Table 3) revealed, however, some rather interesting points.
51
52
A. VAN TIENHOVEN, R. G. D. STEEL AND S. A. DUCHAINE
SUMMARY
Semen diluted 1:15 with milk or Tyrode solution with and without glycine and antibiotics was tested for fertilizing capacity as well as for maintenance of livability during storage at 5°C. Addition of either antibiotics or glycine resulted in higher fertility levels when added to the Tyrode, but when added to milk decreased fertility. The combination of glycine and antibiotics was even more harmful when added to milk, whereas, in the Tyrode solution addition of both antibiotics and glycine had no effect. No reliable prediction of the perform-
with and without glycerol. J. Dairy Sci. 39: 1690-1696. Frobisher, M., 1953. Fundamentals of Microbiology, 5th Ed., W. B. Saunders Co., Philadelphia. Knodt, C. B., and G. W. Salisbury, 1946. The effect of sulfanilamide upon the livability and metabolism of bovine spermatozoa. J. Dairy Sci. 29: 285-291. Kosin, I. L., and A. Wheeler, 1956. Methods for estimating spermatozoal numbers in turkey semen. Northwest Sci. 30: 41—47. Lake, P. E., 1954. The relationship between morphology and function in fowl spermatozoa. Proc. Tenth World's Poultry Congr. 2: 79-85. Lorenz, F. W., and A. Tyler, 1951. Extension of motile life span of spermatozoa of the domestic fowl by amino acids and proteins. Proc. Soc. Exp. Biol. Med. 78: 57-62.
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seems definitely desirable. In vitro glycine ance of a diluent with respect to supporthad a small but significant effect on the ing fertilizing capacity of the sperm can rate of motility. be made with our methods from in vitro Antibiotics had a beneficial effect on studies in which livability and metabolic fertility when added to Tyrode but in- activity are studied before and after storhibitory when added to milk diluents. The age. reason for this effect is not clear at all, A Tyrode solution with added antiespecially because no detrimental effect biotics or glycine give the best fertility, was observed with the milk antibiotic although the fertility level obtained is diluent in the in vitro study. still about 15% below that obtained with On the basis of experiments with cock undiluted semen. semen it seems desirable to use antibiotics ACKNOWLEDGMENTS in the diluents because they inhibit Thanks are due to Mr. J. Zagata for his bacterial growth without affecting semen valuable help in the experiments. livability (Smith, 1949; Terbush, 1952). REFERENCES The present methods required for collection of avian semen make it practically Almquist, J. 0., R. J. Flipse and D. L. Thacker, 1954. Diluters for bovine semen. IV. Fertility of impossible to collect semen without bacbovine spermatozoa in heated homogenized milk terial contamination. In any efforts to and skimmilk. J. Dairy Sci. 37: 1303-1307. find a diluent in which semen can be Bade, M. L., H. Wiegers and L. Nelson, 1956. Oxystored, bacterial growth should be kept gen uptake, motility and fructolysis of turkey at a minimum to obtain better results spermatozoa. J. App. Physiol. 9: 91-96. during storage and also to prevent the Bogdonoff, P. D., Jr., and C. S. Shaffner, 1954. The effect of pH on in vitro survival, metabolic acintroduction of large numbers of bacteria tivity and fertilizing capacity of chicken semen. into the oviduct. Poultry Sci. 33: 665-669. In both groups of diluents the addition Campbell, R. C , H. M. Dott and T. D. Glover, 1956. Nigrosin-eosin as a stain for differentiating of both glycine and antibiotics resulted in live and dead spermatozoa. J. Agr. Sci. 48:1-8. poorer fertility than when either was Flipse, R. J., and J. O. Almquist, 1956. Diluters for added alone. Again we have raised more bovine semen. IX. Motility of bovine spermatoquestions than we have provided answers. zoa in milk-glycine and egg yolk-glycine diluents
DILUENTS FOR TURKEY SEMEN
Snedecor, G., 1956. Statistical Methods, 5th Ed., Iowa State College Press, Ames, Iowa. Swanson, E. W., and H. J. Beardon, 1951. An eosinnigrosin stain for differentiating live and dead bovine spermatozoa. J. An. Sci. 10: 981-987. Terbush, E. L., 1952. The influence of diluent, dilution rate and antibiotic level upon sperm motility and bacterial numbers of fowl semen. M.S. Thesis, Indiana. van Tienhoven, A., and R. G. D. Steel, 1957. The effect of different diluents and dilution rates on fertilizing capacity of turkey semen. Poultry Sci. 36: 473^79.
Air Sacs in the Turkey R. H. RIGDON, T. M. FERGUSON, G. L. FELDMAN AND J. R. COUCH
Laboratory of Experimental Pathology, The University of Texas Medical Branch, Galveston, Texas and Department of Poultry Science, Agricultural and Mechanical College of Texas, College Station, Texas (Received for publication July 3, 1957)
O
NLY one specific study on the respiratory system of the turkey has been found. This consisted of two papers published in 1953 by Cover. Cover reviewed the observations of previous investigators on the chicken. He pointed out that in general all reports agree on the number of air sacs in the chicken (4 paired and 1 single) but they often have been named differently. Disagreement centers around the cervical or thoraco-cervical and the posterior thoracic air sacs. Cover (1953b) observed in the turkey that "the paired posterior thoracic and thoraco-cervical as well as the single anterior thoracic air sacs are combined into a single large compartment, the aggregate sac, which communicates with the air passageways of This investigation was supported by research grants C-1469 (4C) from the National Cancer Institute, and B-759 (C2) from the National Institute of Neurological Diseases and Blindness, of the National Institutes of Health, Public Health Service.
the lung at its anterior ventral border. There are two unions, one with a ventral bronchus and another with an area of several parabronchi. The combined sacs have the same location and visceral relations as the individual sacs which were described by McLeod and Wagers (1939). In addition, however, numerous diverticula are present." The axillary diverticulum in the chicken, according to McLeod and Wagers (1939) "is a large pouch leading from the main sac behind the coracoid bone into the axillary region, where it is covered by the large, superficial pectoral muscle." According to Bradley (1951), the axillary sac in the chicken is a prolongation of the clavicular sac and does not communicate with the bronchi. Our interest in the structure of the air sacs of the turkey arose after finding large hernias in the axilla in sixteen out of twenty Broad Breasted Bronze poults, five weeks of age (Rigdon
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
Phillips, H. J., and H. L. Wiegers, 1952. Settling rate of turkey sperm on a haemocytometer. Poultry Sci. 31:681-684. Qureshi, S. H., 1952. The effects of antibiotics alone and with various diluents and hormones upon in vitro survival and fertilizing capacity of chicken semen. Ph.D. Thesis, Maryland. Salisbury, G. W., G. H. Beck, I. Elliott and E. L. Willett, 1943. Rapid methods for estimating the number of spermatozoa in bull semen. J. Dairy Sci. 26: 69-78. Smith, A. U., 1949. The control of bacterial growth in fowl semen. J. Agr. Sci. 39: 194-200.
53
HERE are, to our knowledge, no reports in published literature on the effect of antibiotics and glycine on turkey spermatozoa. The reports on the effect of these additions to avian semen have been concerned with rooster semen (Smith, 1949; Lorenz and Tyler, 1951; Terbush, 1952; Qureshi, 1952). Van Tienhoven and Steel (1957) reported on experiments with turkey semen diluted with three different diluents at dilution rates ranging from 1:1 to 1:15 in which no differences among dilution rates were detected. However, it has been possible to obtain fertility at higher dilution rates with turkey than with rooster semen (van Tienhoven, unpublished), thus indicating differences between semen of these species.
In the fertility experiment 117 hens were distributed at random over 13 pens of 9 hens each. Within each pen each treatment was represented once and the hens were assigned at random to the treatments. The random assignments were used because no prediction could be made of a particular hen's expected fertility. Inseminations in pens 2-12 were made on February 4, 6 and 25 while in pens 13 and 14, which came into production later, the dates of insemination were February 14 and 25. Three hundredths of a c.c. of either diluted or undiluted semen was inseminated. We determined fertility according to the methods outlined previously (van Tienhoven and Steel, 1957). The maximum length of storage of eggs was 10 days.
We decided to study the effect of added glycine and antibiotics on both fertilizing capacity and livability of turkey semen in different diluents using dilution rates of 1:15.
Sperm concentration determinations were made according to the method of Salisbury el al. (1943). The optical density of semen diluted 1:500 was determined at a wavelength of 600 ran. In our laboratory the calibration curve for this procedure was made with the aid of hemocytometer counts according to Phillips and Wiegers (1952). The regression found can be expressed by the equation:
MATERIALS AND METHODS In all experiments semen from 15-20 toms was pooled and the semen diluted 1:15 with each of the diluents in Table 1. Dilutions were completed within 1 hour after the start of collection and the inseminations were completed within 70 minutes after dilution. Motility examinations and other laboratory procedures necessary for the determinations were finished within 30 minutes after dilution. 1
2
Department of Poultry Husbandry.
Department of Plant Breeding, Biometrics Unit.
F=1984.6X-141.2; r = 0.865 In which F = sperm concentration X 10,000/mm3 X = optical density at 600 rafi r = correlation coefficient The regression coefficient was highly significant (P<0.01). The work of Kosin and Wheeler (1956) shows that estimates made according to the method of Phillips
47
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
T
48
A. VAN TIENHOVEN, R. G. D. STEEL AND S. A. DUCHAINE TABLE 1.—Composition of diluents used in experiments 1 and 2 Constituents
No.
Tyrode,* Milkf parts
Tyrode (T) Tyrode+Glycine (TG) Tyrode+Antibiotics (TA) Tyrode+Glycine+Antibiotics (TGA) Milk (M) Milk+Glycine (MG) Milk+Antibiotics (MA) Milk+Glycine+Antibiotics (MGA)
Clvcine i P e n ' c il" Strep to- SulfanilIA7 ™ lin> mycin, amide, units cart?' / mcS-/ m§-/ ^ ml. ml. ml.
LUU
31 100 31
— — — —
.
19
— —.
100 31 100 31
— 19
1,000 1,000
20 20
3 3
—
—. —.
— —.
— —
19
— 19
1,000 1,000
20 20
3 3
* Tyrode according to Bogdonoff and Shaffner (1954) p H = 6 . 8 . t Milk according to Almquist et al. (1954) p H = 6 . 7 . t Glycine 0.167 m. is isotonic with the Tyrode solution used.
and Wiegers (1952) may be too low. This publication came to our attention after the work on this experiment was completed. Methylene blue reduction time (MBRT) and motility were determined according to methods described previously (van Tienhoven and Steel, 1957). For the dead sperm count a total of 400 sperm were counted on each of duplicate slides, while in the motility determinations an incubation stage was used. For the dead sperm count 1 drop of the diluted semen was mixed with about 10 drops of a nigrosin-eosin stain, consisting of 5% nigrosin and 1% eosin dissolved in Tyrode solution. The sperm were exposed to the stain for two minutes and the smear made by pulling two slides apart. The slides were immediately dried by passing them through a flame and by gentle blowing. This method is an adaptation of the methods used for bull sperm by Swanson and Bearden (1951) and by Bade et al. (1956) for turkey sperm. A total of 400 cells was counted on each slide and precautions were taken that the results obtained on one slide were unknown to the observer while the replicate slide was
counted. Campbell et al. (1956) have shown that knowledge of the count on one slide may introduce bias when the second count is made. All counts were made by one person (T.N.). 1 EXPERIMENTS AND RESULTS
Experiment 1: We studied the fertilizing capacity of the semen after dilution with each of the diluents given in Table 1. The concentrations of streptomycin and penicillin are the same as used by Terbush (1952). The sulfanilamide concentration is the same as used for bull semen (Knodt and Salisbury, 1946). The number of sperm inseminated varied on the different insemination dates between 10.34-14.79 million sperm per insemination. The results of the experiment are presented in Table 2. Although 13 hens were inseminated on each treatment, some failed to lay in the trapnest. This explains the variation in the number of hens inseminated in each treatment group. Because of the rather high incidence of hens laying infertile eggs in some treatments, we decided to test whether the 1 Thanks are due to Mr. T. Noszlopi for his help in these experiments.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on November 22, 2014
1 2 3 4 5 6 7 8
Description
49
DILUENTS FOR TURKEY SEMEN TABLE 2.—The effect of diluents upon fertilizing ca-
pacity of turkey semen Eggs Diluent
Fertility Percent
257 277 223 234 261 208 267 264 230
57.6 67.9 77.1 50.4 52.9 21.6 28.8 15.5 89.7
T TG TA TGA M MG MA MGA Undil.
— Hens inseminated 12 13 13 11 13 13 13 13 11
infertility was due to the treatment or to inherent infertility of the hens. All these hens were subsequently inseminated with undiluted semen and those from which eggs were obtained proved to be fertile. We therefore included all hens in the analysis of the data. The missing data, due to failure to lay in the nest, broodiness, etc., made an analysis of variance of the percentage fertile eggs cumbersome. We therefore decided to analyze the data by a series of chi square tests (Snedecor, 1956) rather than by an analysis of variance. Some biological interactions are so clearcut that no tests of significance were made.
The fertility trial shows clearly that: 1) The differences between the milk and Tyrode diluents are small when no glycine or antibiotics are added. Although the fertility levels obtained were lower than in our previ-
TABLE 3.—The effect of various diluents on semen livability and metabolic activity Day 2
Day 1 Diluent
T TXG TXA
TXGXA M MXG MXA
MXGXA Tvs. M G
(T-M)G (T-M) A GXA (T-M) GXA TXGXA MXGXA Samples
MBRT, nun.
Dead/ - 400 Rate counted
Motility
%
MBRT, mm.
Day 3
Motility
%
Dead/ • 400 Rate counted
Motility
MBRT, mm.
%
Rate 0
4.00 3.75 3.21 3.71
65.0 56.7 70.0 51.7
3.40 3.25 3.25 3.09
80.9 80.4 95.3 75.4
33.25 16.29 15.25 32.89
10.8 16.7 16.7
1.10 1.16 1.33 1.00
102.8 134.5 146.3 142.6
55.5 47.4 52.0 54.3
0 1 1.4 0
4.41 5.13 5.80 5.77
56.7 55.0 53.3 51.7
3.33 3.40 3.00 3.25
39.3 36.1 39.8 47.7
11.32 11.19 16.63 10.56
9.2
1.00 1.83 1.16 1.92
88.7 141.1 117.4 137.3
22.2 20.5 40.7 32.55
1 1 0 5
0
NS NS NS NS NS NS NS
<0.01
<0.01
NS NS NS NS NS NS
NS NS NS NS NS NS NS
NS NS NS NS NS NS
NS NS NS NS
NS NS NS NS NS NS NS
<0.01
<0.05
<0.01
NS
<0.01
<0.01
<0.05 <0.01 <0.01 <0.05 <0.01
8.3
11.7 15.0 20.0
<0.01
NS NS NS NS
NS NS NS NS NS NS NS
<0.05 NS NS NS
NS NS NS NS NS NS NS
<0.01
NS
<0.01
<0.01
NS NS NS NS NS NS NS
<0.05 <0.05 <0.05
<0.01
NS NS
.2 .5
— .3 .3 .8
-
Dead/ 400 counted 210.3 108.5 179.5 133.9 157.0 224.4 231.0 153.0 NS
<0.05 NS NS NS NS NS
NS
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Set
The results posed the question whether it would be feasible to predict the performance of sperm in certain diluents, with respect to fertilizing capacity, from the ability of the diluent to support sperm activity and livability in vitro. The advantages of such a preliminary screening of diluents by in vitro studies are selfevident. The large differences between diluents in this experiment suggested the use of the same diluents for in vitro studies. Experiment 2: After the semen was diluted 1:15 with the various diluents, MBRT, motility and incidence of dead sperm were determined. The semen was subsequently stored at 5°C. and the same determinations made 24 and 48 hours later. The results are given in Table 3. Analyses of variance for the various characteristics studied were computed separately for Day 1, 2, and 3 partly because of heterogeneity of variance. DISCUSSION
50
A. VAN TIENHOVEN, R. G. D. STEEL AND S. A. DUCHAINE
4) Although the T.A. diluent gave better results than the other diluents, the fertility level obtained with this diluent is lower than that obtained with undiluted semen. 5) In the Tyrode solution there was no decline in fertility with increasing
hatch number, but with milk diluents the fertility declined in spite of inseminations repeated three weeks after the first insemination. This strongly suggests that the milk diluent may create a condition in the oviduct unfavorable to fertilization by other sperm also diluted with milk. We have not investigated whether this would also hold true if undiluted semen were used for the later inseminations. 6) The differences between hatches were highly significant, probably due mainly to the effect discussed under 5. 7) The differences between pens are highly significant even when account is taken of the fact that in some pens more birds failed to lay than in others. The data are given in Table 4. We cannot discover any physiological reason why there is a pen effect on fertility. In our previous work we showed that the time between collection and insemination had little or no effect on subsequent fertility. In the present experiments care was taken that the order in TABLE 4.—The effect of pen on fertilizing capacity of turkey semen Eggs
2 3 4 5 6 7 8 9 10 11 12 13 14
Set
Fertile
no. 133 202 230 215 124 209 171 162 166 216 187 105 101
61.7 60.0 36.1 48.4 62.1 55.0 62.0 53.7 29.5 51.4 47.6 70.5 52.5
%
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ous experiment (van Tienhoven and Steel, 1957), the comparison did not lead to different conclusions. 2) The addition of antibiotics to Tyrode diluent caused increased fertility but when added to milk it caused a decrease. This interaction of diluents and antibiotics i.e. the failure of the addition of antibiotics to the two diluents to produce the same change in fertility, was highly significant (<0.01). Similarly, glycine addition to the Tyrode diluent resulted in better fertility than obtained with Tyrode alone, but when glycine was added to milk it had a deleterious effect on fertilizing capacity. This diluent glycine interaction was highly significant ( P < 0.01). 3) The combination of glycine and antibiotics added to Tyrode solution resulted in a fertility not significantly different from the fertility obtained with semen diluted with Tyrode solution. The combination of glycine and antibiotics added to milk resulted in a further depression of fertility, below that found with the milk-glycine or milk-antibiotics diluents. The interaction of diluents and antibiotics plus glycine was highly significant (P<0.01). The three-way interaction implied by this paragraph as related to discussion item 2 was not tested for significance because the magnitudes of the differences involved made such a calculation superfluous.
DILUENTS FOR TURKEY SEMEN
which the pens were inseminated was rotated.
1) On Day 1 the semen diluted with milk diluents had a longer MBRT than semen diluted with Tyrode diluents. This difference is small and may well be due to the fact that the end point in the milk diluent is not as sharp as in the Tyrode solution. 2) The incidence of dead sperm in milk diluents on Day 1 is smaller than in the Tyrode diluents. This difference was not due to interference of the milk with the staining procedure as such. This was demonstrated for semen diluted with Tyrode; just before the stain was added, one or two drops of milk were added and the semen processed as usual. The same was done for semen diluted with milk and subsequent addition of Tyrode just before staining. The results obtained with the Tyrode and milk diluent seem to indicate that the dilution with milk poses less of a "shock" to the sperm than the dilution with Tyrode diluents. However, on further storage there is no significant difference between the diluents.
3) The MBRT is considerably shorter in milk than in Tyrode diluents on Day 2. Although we have not made any bacterial counts in this study, it seems unlikely that the difference between the two diluents is due to greater bacterial growth in the milk diluent. If such were the case antibiotics which suppress bacterial growth (Smith, 1949) might be expected to affect MBRT because the latter is affected by the presence of bacteria (Frobisher, 1953). As antibiotics did not affect MBRT we conclude, tentatively, that milk diluents support metabolic activity better than Tyrode solution, without affecting livability, as can be seen from the incidence of dead sperm in the two groups of diluents. The addition of glycine, which has been reported to be beneficial in supporting the livability of cock semen at room temperature (Lorenz and Tyler, 1951) and of bull semen at 5°C. (Flipse and Almquist, 1956) has a beneficial effect on fertility when added to Tyrode solution but an inhibitory effect when added to milk. Flipse and Almquist (1956) showed that small amounts of glycine actually are harmful in supporting livability. The concentrations which were beneficial in the trials with bull semen (Flipse and Almquist, 1956) are hypertonic. For fowl semen, at least, the work by Lake (1954) indicates that isotonicity of the diluent may be particularly important. For this reason the concentrations used by us are considerably lower than those used by Flipse and Almquist (1956) but the concentrations are the same as those found to be most beneficial for fowl semen by Lorenz and Tyler (1951). A further study of this phenomenon of a beneficial effect of a hypertonic solution
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A comparison between the results of Experiments 1 and 2 shows that with these diluents, at least, the screening of diluents by in vitro studies was not successful. It is, of course, possible that a diluent which supports livability exceptionally well could be screened out this way and then used for fertility trials. But relatively minor differences or even the rather large differences as found on Day 2 in MBRT (see Table 3) do not give any lead to the future success of a diluent in a fertility trial. The in vitro study (see Table 3) revealed, however, some rather interesting points.
51
52
A. VAN TIENHOVEN, R. G. D. STEEL AND S. A. DUCHAINE
SUMMARY
Semen diluted 1:15 with milk or Tyrode solution with and without glycine and antibiotics was tested for fertilizing capacity as well as for maintenance of livability during storage at 5°C. Addition of either antibiotics or glycine resulted in higher fertility levels when added to the Tyrode, but when added to milk decreased fertility. The combination of glycine and antibiotics was even more harmful when added to milk, whereas, in the Tyrode solution addition of both antibiotics and glycine had no effect. No reliable prediction of the perform-
with and without glycerol. J. Dairy Sci. 39: 1690-1696. Frobisher, M., 1953. Fundamentals of Microbiology, 5th Ed., W. B. Saunders Co., Philadelphia. Knodt, C. B., and G. W. Salisbury, 1946. The effect of sulfanilamide upon the livability and metabolism of bovine spermatozoa. J. Dairy Sci. 29: 285-291. Kosin, I. L., and A. Wheeler, 1956. Methods for estimating spermatozoal numbers in turkey semen. Northwest Sci. 30: 41—47. Lake, P. E., 1954. The relationship between morphology and function in fowl spermatozoa. Proc. Tenth World's Poultry Congr. 2: 79-85. Lorenz, F. W., and A. Tyler, 1951. Extension of motile life span of spermatozoa of the domestic fowl by amino acids and proteins. Proc. Soc. Exp. Biol. Med. 78: 57-62.
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seems definitely desirable. In vitro glycine ance of a diluent with respect to supporthad a small but significant effect on the ing fertilizing capacity of the sperm can rate of motility. be made with our methods from in vitro Antibiotics had a beneficial effect on studies in which livability and metabolic fertility when added to Tyrode but in- activity are studied before and after storhibitory when added to milk diluents. The age. reason for this effect is not clear at all, A Tyrode solution with added antiespecially because no detrimental effect biotics or glycine give the best fertility, was observed with the milk antibiotic although the fertility level obtained is diluent in the in vitro study. still about 15% below that obtained with On the basis of experiments with cock undiluted semen. semen it seems desirable to use antibiotics ACKNOWLEDGMENTS in the diluents because they inhibit Thanks are due to Mr. J. Zagata for his bacterial growth without affecting semen valuable help in the experiments. livability (Smith, 1949; Terbush, 1952). REFERENCES The present methods required for collection of avian semen make it practically Almquist, J. 0., R. J. Flipse and D. L. Thacker, 1954. Diluters for bovine semen. IV. Fertility of impossible to collect semen without bacbovine spermatozoa in heated homogenized milk terial contamination. In any efforts to and skimmilk. J. Dairy Sci. 37: 1303-1307. find a diluent in which semen can be Bade, M. L., H. Wiegers and L. Nelson, 1956. Oxystored, bacterial growth should be kept gen uptake, motility and fructolysis of turkey at a minimum to obtain better results spermatozoa. J. App. Physiol. 9: 91-96. during storage and also to prevent the Bogdonoff, P. D., Jr., and C. S. Shaffner, 1954. The effect of pH on in vitro survival, metabolic acintroduction of large numbers of bacteria tivity and fertilizing capacity of chicken semen. into the oviduct. Poultry Sci. 33: 665-669. In both groups of diluents the addition Campbell, R. C , H. M. Dott and T. D. Glover, 1956. Nigrosin-eosin as a stain for differentiating of both glycine and antibiotics resulted in live and dead spermatozoa. J. Agr. Sci. 48:1-8. poorer fertility than when either was Flipse, R. J., and J. O. Almquist, 1956. Diluters for added alone. Again we have raised more bovine semen. IX. Motility of bovine spermatoquestions than we have provided answers. zoa in milk-glycine and egg yolk-glycine diluents
DILUENTS FOR TURKEY SEMEN
Snedecor, G., 1956. Statistical Methods, 5th Ed., Iowa State College Press, Ames, Iowa. Swanson, E. W., and H. J. Beardon, 1951. An eosinnigrosin stain for differentiating live and dead bovine spermatozoa. J. An. Sci. 10: 981-987. Terbush, E. L., 1952. The influence of diluent, dilution rate and antibiotic level upon sperm motility and bacterial numbers of fowl semen. M.S. Thesis, Indiana. van Tienhoven, A., and R. G. D. Steel, 1957. The effect of different diluents and dilution rates on fertilizing capacity of turkey semen. Poultry Sci. 36: 473^79.
Air Sacs in the Turkey R. H. RIGDON, T. M. FERGUSON, G. L. FELDMAN AND J. R. COUCH
Laboratory of Experimental Pathology, The University of Texas Medical Branch, Galveston, Texas and Department of Poultry Science, Agricultural and Mechanical College of Texas, College Station, Texas (Received for publication July 3, 1957)
O
NLY one specific study on the respiratory system of the turkey has been found. This consisted of two papers published in 1953 by Cover. Cover reviewed the observations of previous investigators on the chicken. He pointed out that in general all reports agree on the number of air sacs in the chicken (4 paired and 1 single) but they often have been named differently. Disagreement centers around the cervical or thoraco-cervical and the posterior thoracic air sacs. Cover (1953b) observed in the turkey that "the paired posterior thoracic and thoraco-cervical as well as the single anterior thoracic air sacs are combined into a single large compartment, the aggregate sac, which communicates with the air passageways of This investigation was supported by research grants C-1469 (4C) from the National Cancer Institute, and B-759 (C2) from the National Institute of Neurological Diseases and Blindness, of the National Institutes of Health, Public Health Service.
the lung at its anterior ventral border. There are two unions, one with a ventral bronchus and another with an area of several parabronchi. The combined sacs have the same location and visceral relations as the individual sacs which were described by McLeod and Wagers (1939). In addition, however, numerous diverticula are present." The axillary diverticulum in the chicken, according to McLeod and Wagers (1939) "is a large pouch leading from the main sac behind the coracoid bone into the axillary region, where it is covered by the large, superficial pectoral muscle." According to Bradley (1951), the axillary sac in the chicken is a prolongation of the clavicular sac and does not communicate with the bronchi. Our interest in the structure of the air sacs of the turkey arose after finding large hernias in the axilla in sixteen out of twenty Broad Breasted Bronze poults, five weeks of age (Rigdon
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Phillips, H. J., and H. L. Wiegers, 1952. Settling rate of turkey sperm on a haemocytometer. Poultry Sci. 31:681-684. Qureshi, S. H., 1952. The effects of antibiotics alone and with various diluents and hormones upon in vitro survival and fertilizing capacity of chicken semen. Ph.D. Thesis, Maryland. Salisbury, G. W., G. H. Beck, I. Elliott and E. L. Willett, 1943. Rapid methods for estimating the number of spermatozoa in bull semen. J. Dairy Sci. 26: 69-78. Smith, A. U., 1949. The control of bacterial growth in fowl semen. J. Agr. Sci. 39: 194-200.
53