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Recovery of Spermatozoa from the Reproductive Tract of Turkey Hens1
Recovery of Spermatozoa from the Reproductive Tract of Turkey Hens 1 O. P. VERMA AND F. L. CHERMS Department of Poultry Science, University of Wisconsin, Madison, Wisconsin (Received for publication November 20, 1965)
V
Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, College of Agriculture, Madison. 1 Supported by grants from the National Turkey Federation and the Wisconsin Turkey Federation.
604
from the oviduct. This paper describes a method of recovering sperm cells from the reproductive tract of turkeys and a comparison of the number of cells recovered from various segments of the oviduct. MATERIALS AND METHODS
Broad Breasted Bronze turkey hens were inseminated weekly with 1/40 c.c. of pooled semen collected with an aspirator kept at a temperature of 20° to 21°C. The inseminations were made as soon as possible after the collection of the semen. The birds were killed on the third day following insemination with an overdose of 'pentobarbital sodium.' For the purpose of this study three sections of the oviduct were studied: the infundibulum, the infundibulum-magnum junction, and the uterus including the uterovaginal junction. The carcass was opened and one section of the oviduct was removed at a time, leaving the remainder within the body cavity. Two washings with 5 c.c. of 0.9% saline solution kept at 41° to 43.5°C. were made on each segment. The infundibulum was removed first and saline was forced through the posterior end and the washings were collected at the anterior end. The infundibulum-magnum junction and the uterus were tied at both ends and saline was injected through the muscular wall. After a thorough mixing, one of the ends was untied and the washings were collected. The recovery of the washings was 8-9 c.c. from the infundibulum and the infundibulum-magnum junction and 6-8 c.c. from the uterus. The washings were centrifuged at 3,000 r.p.m. for 25 minutes and
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
INTRODUCTION ARIOUS attempts have been made by several workers using different procedures to recover spermatozoa from the oviduct of chickens. Payne (1914) collected the scrapings from the different parts of the oviduct and recovered tailless sperm from 2 to 56 days after separation of hens from the roosters. Walton and Whetham (1933) were unable to find spermatozoa in the oviduct of the hens from 1 to IS days after separation from the cocks. Warren and Kilpatrick (1929) placed small amounts of normal saline solution on the mucous membrane of the oviduct and collected a sample with a platinum loop. They recovered sperm from 6 hours to 18 days after natural mating, most of which had lost their flagella. Van Drimmelen (1945) added Ringer's physiological saline solution on the inner surface of the oviduct with the help of a fine capillary glass tube and collected the material with the same tube. He found complete sperm with tail from 3 to 14 days following insemination. Grigg (1957) passed an artificial sac of cellophane filled with Ringer's solution through the infundibulum and recovered numerous sperm cells from the surface of the sac with intact tail after three hours following insemination. In turkey hens, however, no reports are available on the recovery of spermatozoa
60S
SPERM RECOVERY I N TURKEYS
TABLE 1.—Number of sperm cells counted in one
cubic millimeter of washings of three sections of the turkey oviduct Infundibulum A1 B2
100 55 105 135 92 127 75
52 32 25 107
InfundibulumMagnum A B
— — —
—
— —
67 117 142 72 110
98
60
101
77 67
72 22 40 110 47 40
A
22
105 120 197 550 135 92 72 322
50
199
—
Uterus B 57 25 25 150
67 100
— 45
66
1 A — O v i d u c t w i t h an egg in t h e infundibulum or magnum. 2 B - -Oviduct w i t h o u t an egg.
TABLE 2.—Analysis of variance of the number of
sperms in washings from three sections of the turkey oviduct Source Segments Group Groups Xsegments Error Total
df
ss
ras
F
2 1 3 33
35,306 64,803 113,169 116,240
17,653 64,803 37,723 3,522
5.01* 18.39** 10.71**
39
329,518
* Significant at 5% level. ** Significant at i% level.
sections were cut at seven microns and stained by hematoxylin and eosin. RESULTS AND DISCUSSION
The number of spermatozoa in the washings collected from the three segments of the oviduct is shown in Table 1. Due to the presence of numerous cilia, mucous and blood cells some difficulty was encountered in counting the sperm cells. It was not possible to differentiate between the 'live' and 'dead' sperm because of "Brownian Movement" in most of the samples. Some sperm cells were lost since the clear supernatant was found to contain some cells but there were too few to obtain a reliable count on a hemocytometer. The data in Table 1 was analyzed statistically after Steel and Torrie (1960). The analysis of variance (Table 2) showed a significant interaction between segments and groups indicating, that the two factors are not independent of each other. The data were further analyzed by Tukey's test and the number of sperm cells in the uterus of group A was found to be significantly higher than those of any other group or the segment. This part of the oviduct is in the immediate vicinity of the sperm storage area—the uterovaginal junction of the turkey hens (Verma and Cherms, 1964). The significant higher number of sperm cells in the uterus of group A suggest that the evacuation of the sperm cells is enhanced after ovulation. This substantiates the postulation of Verma and Cherms (1965) that there are
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
sufficient supernatant was discarded to leave 1 c.c. of the sample which was then thoroughly mixed. Sperm cell concentration was determined in each sample with a hemocytometer in the same manner by which mammalian white blood cell counts are made. Four large corner squares of the counting chamber, with a total of 64 small squares, were counted. To study the morphology of the recovered sperm, a smear was made with a platinum loop, dried and stained with crystal violet. For further comparison, the birds used in this study were divided into two groups. Birds of the group A had just ovulated and had an egg in either the infundibulum or in the magnum. Group B contained hens that had laid but had not ovulated on the day they were sacrificed. The birds were trapnested and eggs incubated for 4 or S days. All candled infertile eggs were broken out to separate the early deads from infertiles. The uterovaginal junction from birds under study were prepared for histological examination to study the presence of sperm nests. The tissues were fixed in 10% formalin, dehydrated and cleared by an Autotechnicon and embedded in paraffin. The
606
O. P. VERMA AND F. L. CHERMS
TABLE 3.—Individual hen fertility during the last two weeks and the presence of spermatozoa in the nests and lumen of the oviduct Recovered Sperm Bird
Inf.
00.0 00.0 00.0 00.0
No No Yes Yes
32 25 107
30.0 50.0 66.6 75.0
Yes No Yes Yes
52 135 100 105
100.0 100.0 100.0 100.0 100.0 100.0 100.0
Yes Yes Yes Yes Yes Yes Yes
55
Fertility last two weeks
603 608 604 602
0/8 0/8 0/4 0/5
611 605 616 694
3/10
672 606 610 620 619 601 617
2/4
8/12 6/8
9/9 8/8
10/10 7/7 6/6 6/6 5/5
— 67 92 127 75 77
Inf.Mag.
Uterus
22 22 40 110
45 25 25 150
72 67
57 550 105 197
— — 110
—
117 142 72 47
120 322 100 135 92 72 65
other factors, besides a mechanical one, responsible for the evacuation of the sperm nests of the turkey hens. Fertility for the last two weeks before sacrificing the hens and the presence of sperm nests is shown in Table 3. Hens 603, 608, 604, and 611 were studied at the outset of the experiment when there was a serious fertility problem in the flock from which these birds were taken. The first three hens in this group showed a fertility of 0% and the number of sperm cells found in the washings of their lumen were considerably lower than any other hen. The presence of sperm nests was invariably observed in the hens with a higher fertility. One of the hens with 50% fertility did not show the presence of sperm nests, although the maximum number of sperm cells were obtained from the uterine washings. Sperm nests were not observed in two of the hens with 0% fertility, while the remaining two showed the presence of sperm nests. This suggests that the normal storage of sperm cells in the uterovaginal junction of turkey hens does not always result in fertile eggs. The presence of sperm nests in most of the hens studied, indicate that the complete evacuation of sperm nest does not occur with the washing techniques used in this experiment.
SUMMARY
Artificially inseminated Broad Breasted Bronze turkeys were sacrificed on the third day following insemination by an overdose of "Pentobarbital sodium." For the purpose of this study three sections of the ovi-
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
Sperm nests
No.
The stained smears from the sediment of the centrifuged washings showed the presence of spermatozoa with a head, midpiece and a small portion of tail. Complete spermatozoa with tail were observed from the washings of both infundibulum and the uterus of only two hens, 604 and 610. The tailless sperm were not seen to progress by their own movement while the complete sperm cells obtained from these two birds showed a progressive and definite movement. The size of the sperm head was smaller than the normal sperm head of turkeys. This agrees with Payne (1914) who observed tailless sperm cells from the infundibulum which were reduced in size. Warren and Kilkpatrick (1929) observed tailless sperm but they found considerable variation in sperm size not only in the different preparations but also among the individual sperm on a single slide. In chickens, the sperm are stored in the infundibulum (Van Drimmelen, 1946) and also in the uterovaginal junction (Bobr et al., 1962) while in turkey hens the sperm storage area has been reported to be the uterovaginal junction (op cit.) but not the infundibulum. This suggests that the tailless sperm cells have to travel to the infundibulum for each fertilization. Since the motility of the sperm is attributed to its fiagella, it is reasonable to conclude that another mechanism for the sperm travel from the uterus to infundibulum other than its own motility must exist. This has been confirmed by the work of Allen and Grigg (1957) who concluded that the motility of sperm cells that have entered the uterus is of little consequence.
SPERM RECOVERY I N TURKEYS
REFERENCES Allen, T. E., and G. W. Grigg, 1957. Sperm transport in the fowl. Australian J. Agr. Res. 8: 788-799. Bobr, L. W., F. W. Lorenz and F. X. Ogasawara, 1962. The role of uterovaginal junction in the storage of cock spermatozoa. Poultry Sci. 4 1 : 1628. Grigg, G. W., 1957. The structure of the stored sperm in the nature of the release mechanism. Poultry Sci. 36:450-451. Payne, L. F., 1914. Vitality and activity of sperm cells and artificial insemination of the chicken. Oklahoma Agr. Exp. Sta. Cir. 30. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedure of Statistics. McGraw-Hill Book Company. Van Drimmelen, G. C , 1945. The location of spermatozoa in the hen by means of capillary attraction. J. South African Vet. Med. Assoc. 16: 97-101. Van Drimmelen, G. C , 1946. "Sperm nests" in the oviduct of the domestic hens. J. South African Vet. Med. Assoc. 17 : 42-52. Verma, O. P., and F. L. Cherms, 1964. Observations on the oviduct of turkeys. Avian Dis. 8: 19-26. Verma, O. P., and F. L. Cherms, 1965. The appearance of sperm and their persistency in storage tubules of turkey hens after a single insemination. Poultry Sci. 44: 609-613. Walton, A., and E. O. Whetham, 1933. The survival of the spermatozoan of the domestic fowl. J. Expt. Biol. 10: 204-211. Warren, D. C , and L. Kilpatrick, 1929. Fertilization in the domestic fowl. Poultry Sci. 8: 237— 256.
NEWS AND
NOTES
(Continued from page 570) Drs. E. L. Stephenson and D. E. Greene of the Department of Animal Science will direct the research. A $2,000 grant to support research at the University on the effects of blackhead preventive drugs has been made by Whitmoyer Laboratories, Inc., Myerstown, Pa. Dr. P. W. Waldroup has succeeded Dr. Daryle Greene as Assistant Professor of Animal Sciences, University of Arkansas. Dr. Greene resigned, effective January IS, to become Director of Poultry Research for the Ralston Purina Company.
4-H POULTRY WINNERS Each of the six national winners in the 4-H Poultry Awards Programs received a $500 educational scholarship. The presentations were made by A. Heisdorf, President of Heisdorf & Nelson Farms, Inc., at a special luncheon held during the 44th National 4-H Club Congress in Chicago, November 28 to December 2. The National 4-H Poultry Program is conducted by the Cooperative Extension Service and arranged by the National 4-H Service Committee. The winners were: Fred A. Baker, Jr., Brook-
(Continued on page 611)
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
duct were examined: the infundibulum, the infundibulum-magnum junction, and the uterus including utero-vaginal junction. Each segment was washed twice with S c.c. of 0.9% saline held at 41° to 43.5°C. The last two segments were tied at both ends and injected with normal saline through the muscular wall, whereas the infundibulum was washed by forcing the saline through the posterior end and out the anterior end. All washings were centrifuged and sufficient supernatant was discarded to leave 1 c.c, which was then thoroughly mixed and sperm cell concentration determined by a hemocytometer. The spermatozoa in the stained smears from the centrifuged washings were smaller in size than the normal turkey sperm and the flagellum was absent in most of the samples. In two of the hens, washings from the infundibulum and the uterus showed the presence of intact spermatozoa. For further comparisons, the birds used in this study were divided into two groups. Group A had birds with an egg in either the infundibulum or in the magnum, while Group B had hens with no egg in the tract. Analysis of the data indicated that the uterus from birds in Group A contained significantly greater numbers of sperm cells than any other segment in either group.
607
V
Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, College of Agriculture, Madison. 1 Supported by grants from the National Turkey Federation and the Wisconsin Turkey Federation.
604
from the oviduct. This paper describes a method of recovering sperm cells from the reproductive tract of turkeys and a comparison of the number of cells recovered from various segments of the oviduct. MATERIALS AND METHODS
Broad Breasted Bronze turkey hens were inseminated weekly with 1/40 c.c. of pooled semen collected with an aspirator kept at a temperature of 20° to 21°C. The inseminations were made as soon as possible after the collection of the semen. The birds were killed on the third day following insemination with an overdose of 'pentobarbital sodium.' For the purpose of this study three sections of the oviduct were studied: the infundibulum, the infundibulum-magnum junction, and the uterus including the uterovaginal junction. The carcass was opened and one section of the oviduct was removed at a time, leaving the remainder within the body cavity. Two washings with 5 c.c. of 0.9% saline solution kept at 41° to 43.5°C. were made on each segment. The infundibulum was removed first and saline was forced through the posterior end and the washings were collected at the anterior end. The infundibulum-magnum junction and the uterus were tied at both ends and saline was injected through the muscular wall. After a thorough mixing, one of the ends was untied and the washings were collected. The recovery of the washings was 8-9 c.c. from the infundibulum and the infundibulum-magnum junction and 6-8 c.c. from the uterus. The washings were centrifuged at 3,000 r.p.m. for 25 minutes and
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
INTRODUCTION ARIOUS attempts have been made by several workers using different procedures to recover spermatozoa from the oviduct of chickens. Payne (1914) collected the scrapings from the different parts of the oviduct and recovered tailless sperm from 2 to 56 days after separation of hens from the roosters. Walton and Whetham (1933) were unable to find spermatozoa in the oviduct of the hens from 1 to IS days after separation from the cocks. Warren and Kilpatrick (1929) placed small amounts of normal saline solution on the mucous membrane of the oviduct and collected a sample with a platinum loop. They recovered sperm from 6 hours to 18 days after natural mating, most of which had lost their flagella. Van Drimmelen (1945) added Ringer's physiological saline solution on the inner surface of the oviduct with the help of a fine capillary glass tube and collected the material with the same tube. He found complete sperm with tail from 3 to 14 days following insemination. Grigg (1957) passed an artificial sac of cellophane filled with Ringer's solution through the infundibulum and recovered numerous sperm cells from the surface of the sac with intact tail after three hours following insemination. In turkey hens, however, no reports are available on the recovery of spermatozoa
60S
SPERM RECOVERY I N TURKEYS
TABLE 1.—Number of sperm cells counted in one
cubic millimeter of washings of three sections of the turkey oviduct Infundibulum A1 B2
100 55 105 135 92 127 75
52 32 25 107
InfundibulumMagnum A B
— — —
—
— —
67 117 142 72 110
98
60
101
77 67
72 22 40 110 47 40
A
22
105 120 197 550 135 92 72 322
50
199
—
Uterus B 57 25 25 150
67 100
— 45
66
1 A — O v i d u c t w i t h an egg in t h e infundibulum or magnum. 2 B - -Oviduct w i t h o u t an egg.
TABLE 2.—Analysis of variance of the number of
sperms in washings from three sections of the turkey oviduct Source Segments Group Groups Xsegments Error Total
df
ss
ras
F
2 1 3 33
35,306 64,803 113,169 116,240
17,653 64,803 37,723 3,522
5.01* 18.39** 10.71**
39
329,518
* Significant at 5% level. ** Significant at i% level.
sections were cut at seven microns and stained by hematoxylin and eosin. RESULTS AND DISCUSSION
The number of spermatozoa in the washings collected from the three segments of the oviduct is shown in Table 1. Due to the presence of numerous cilia, mucous and blood cells some difficulty was encountered in counting the sperm cells. It was not possible to differentiate between the 'live' and 'dead' sperm because of "Brownian Movement" in most of the samples. Some sperm cells were lost since the clear supernatant was found to contain some cells but there were too few to obtain a reliable count on a hemocytometer. The data in Table 1 was analyzed statistically after Steel and Torrie (1960). The analysis of variance (Table 2) showed a significant interaction between segments and groups indicating, that the two factors are not independent of each other. The data were further analyzed by Tukey's test and the number of sperm cells in the uterus of group A was found to be significantly higher than those of any other group or the segment. This part of the oviduct is in the immediate vicinity of the sperm storage area—the uterovaginal junction of the turkey hens (Verma and Cherms, 1964). The significant higher number of sperm cells in the uterus of group A suggest that the evacuation of the sperm cells is enhanced after ovulation. This substantiates the postulation of Verma and Cherms (1965) that there are
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
sufficient supernatant was discarded to leave 1 c.c. of the sample which was then thoroughly mixed. Sperm cell concentration was determined in each sample with a hemocytometer in the same manner by which mammalian white blood cell counts are made. Four large corner squares of the counting chamber, with a total of 64 small squares, were counted. To study the morphology of the recovered sperm, a smear was made with a platinum loop, dried and stained with crystal violet. For further comparison, the birds used in this study were divided into two groups. Birds of the group A had just ovulated and had an egg in either the infundibulum or in the magnum. Group B contained hens that had laid but had not ovulated on the day they were sacrificed. The birds were trapnested and eggs incubated for 4 or S days. All candled infertile eggs were broken out to separate the early deads from infertiles. The uterovaginal junction from birds under study were prepared for histological examination to study the presence of sperm nests. The tissues were fixed in 10% formalin, dehydrated and cleared by an Autotechnicon and embedded in paraffin. The
606
O. P. VERMA AND F. L. CHERMS
TABLE 3.—Individual hen fertility during the last two weeks and the presence of spermatozoa in the nests and lumen of the oviduct Recovered Sperm Bird
Inf.
00.0 00.0 00.0 00.0
No No Yes Yes
32 25 107
30.0 50.0 66.6 75.0
Yes No Yes Yes
52 135 100 105
100.0 100.0 100.0 100.0 100.0 100.0 100.0
Yes Yes Yes Yes Yes Yes Yes
55
Fertility last two weeks
603 608 604 602
0/8 0/8 0/4 0/5
611 605 616 694
3/10
672 606 610 620 619 601 617
2/4
8/12 6/8
9/9 8/8
10/10 7/7 6/6 6/6 5/5
— 67 92 127 75 77
Inf.Mag.
Uterus
22 22 40 110
45 25 25 150
72 67
57 550 105 197
— — 110
—
117 142 72 47
120 322 100 135 92 72 65
other factors, besides a mechanical one, responsible for the evacuation of the sperm nests of the turkey hens. Fertility for the last two weeks before sacrificing the hens and the presence of sperm nests is shown in Table 3. Hens 603, 608, 604, and 611 were studied at the outset of the experiment when there was a serious fertility problem in the flock from which these birds were taken. The first three hens in this group showed a fertility of 0% and the number of sperm cells found in the washings of their lumen were considerably lower than any other hen. The presence of sperm nests was invariably observed in the hens with a higher fertility. One of the hens with 50% fertility did not show the presence of sperm nests, although the maximum number of sperm cells were obtained from the uterine washings. Sperm nests were not observed in two of the hens with 0% fertility, while the remaining two showed the presence of sperm nests. This suggests that the normal storage of sperm cells in the uterovaginal junction of turkey hens does not always result in fertile eggs. The presence of sperm nests in most of the hens studied, indicate that the complete evacuation of sperm nest does not occur with the washing techniques used in this experiment.
SUMMARY
Artificially inseminated Broad Breasted Bronze turkeys were sacrificed on the third day following insemination by an overdose of "Pentobarbital sodium." For the purpose of this study three sections of the ovi-
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
Sperm nests
No.
The stained smears from the sediment of the centrifuged washings showed the presence of spermatozoa with a head, midpiece and a small portion of tail. Complete spermatozoa with tail were observed from the washings of both infundibulum and the uterus of only two hens, 604 and 610. The tailless sperm were not seen to progress by their own movement while the complete sperm cells obtained from these two birds showed a progressive and definite movement. The size of the sperm head was smaller than the normal sperm head of turkeys. This agrees with Payne (1914) who observed tailless sperm cells from the infundibulum which were reduced in size. Warren and Kilkpatrick (1929) observed tailless sperm but they found considerable variation in sperm size not only in the different preparations but also among the individual sperm on a single slide. In chickens, the sperm are stored in the infundibulum (Van Drimmelen, 1946) and also in the uterovaginal junction (Bobr et al., 1962) while in turkey hens the sperm storage area has been reported to be the uterovaginal junction (op cit.) but not the infundibulum. This suggests that the tailless sperm cells have to travel to the infundibulum for each fertilization. Since the motility of the sperm is attributed to its fiagella, it is reasonable to conclude that another mechanism for the sperm travel from the uterus to infundibulum other than its own motility must exist. This has been confirmed by the work of Allen and Grigg (1957) who concluded that the motility of sperm cells that have entered the uterus is of little consequence.
SPERM RECOVERY I N TURKEYS
REFERENCES Allen, T. E., and G. W. Grigg, 1957. Sperm transport in the fowl. Australian J. Agr. Res. 8: 788-799. Bobr, L. W., F. W. Lorenz and F. X. Ogasawara, 1962. The role of uterovaginal junction in the storage of cock spermatozoa. Poultry Sci. 4 1 : 1628. Grigg, G. W., 1957. The structure of the stored sperm in the nature of the release mechanism. Poultry Sci. 36:450-451. Payne, L. F., 1914. Vitality and activity of sperm cells and artificial insemination of the chicken. Oklahoma Agr. Exp. Sta. Cir. 30. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedure of Statistics. McGraw-Hill Book Company. Van Drimmelen, G. C , 1945. The location of spermatozoa in the hen by means of capillary attraction. J. South African Vet. Med. Assoc. 16: 97-101. Van Drimmelen, G. C , 1946. "Sperm nests" in the oviduct of the domestic hens. J. South African Vet. Med. Assoc. 17 : 42-52. Verma, O. P., and F. L. Cherms, 1964. Observations on the oviduct of turkeys. Avian Dis. 8: 19-26. Verma, O. P., and F. L. Cherms, 1965. The appearance of sperm and their persistency in storage tubules of turkey hens after a single insemination. Poultry Sci. 44: 609-613. Walton, A., and E. O. Whetham, 1933. The survival of the spermatozoan of the domestic fowl. J. Expt. Biol. 10: 204-211. Warren, D. C , and L. Kilpatrick, 1929. Fertilization in the domestic fowl. Poultry Sci. 8: 237— 256.
NEWS AND
NOTES
(Continued from page 570) Drs. E. L. Stephenson and D. E. Greene of the Department of Animal Science will direct the research. A $2,000 grant to support research at the University on the effects of blackhead preventive drugs has been made by Whitmoyer Laboratories, Inc., Myerstown, Pa. Dr. P. W. Waldroup has succeeded Dr. Daryle Greene as Assistant Professor of Animal Sciences, University of Arkansas. Dr. Greene resigned, effective January IS, to become Director of Poultry Research for the Ralston Purina Company.
4-H POULTRY WINNERS Each of the six national winners in the 4-H Poultry Awards Programs received a $500 educational scholarship. The presentations were made by A. Heisdorf, President of Heisdorf & Nelson Farms, Inc., at a special luncheon held during the 44th National 4-H Club Congress in Chicago, November 28 to December 2. The National 4-H Poultry Program is conducted by the Cooperative Extension Service and arranged by the National 4-H Service Committee. The winners were: Fred A. Baker, Jr., Brook-
(Continued on page 611)
Downloaded from http://ps.oxfordjournals.org/ by Nathalia Oliveira on May 10, 2015
duct were examined: the infundibulum, the infundibulum-magnum junction, and the uterus including utero-vaginal junction. Each segment was washed twice with S c.c. of 0.9% saline held at 41° to 43.5°C. The last two segments were tied at both ends and injected with normal saline through the muscular wall, whereas the infundibulum was washed by forcing the saline through the posterior end and out the anterior end. All washings were centrifuged and sufficient supernatant was discarded to leave 1 c.c, which was then thoroughly mixed and sperm cell concentration determined by a hemocytometer. The spermatozoa in the stained smears from the centrifuged washings were smaller in size than the normal turkey sperm and the flagellum was absent in most of the samples. In two of the hens, washings from the infundibulum and the uterus showed the presence of intact spermatozoa. For further comparisons, the birds used in this study were divided into two groups. Group A had birds with an egg in either the infundibulum or in the magnum, while Group B had hens with no egg in the tract. Analysis of the data indicated that the uterus from birds in Group A contained significantly greater numbers of sperm cells than any other segment in either group.
607