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Fertilization in the Domestic Fowl1
POULTRY SCIENCE (Reg. U. S. Pat. Off.)
VOL. VIIT
JUNE-JULY, 1929
FERTILIZATION IN THE DOMESTIC FOWL
No.~5 1
T H E E F F E C T OF F R E S H N E S S U P O N T H E COMPETITIVE E F F I C I E N C Y OF SPERM
Crew 5 called attention to t h e fact t h a t when one male was r e placed by another, the period t h r o u g h which the sperm of t h e i Contribution No. 46 Department of JPoultry Husbandry. Read at the annual meeting of Poultry Science Association, held at Lafayette, Indiana, August 21-24, 1928. 2 DUNN, L. C, 1927. Selective fertilization in fowls. POULTRY SCIENCE
6:201-214. s CREW, P. A. E., 1926. On fertility in the domestic fowl. Proc. R. Soc. Edinburgh 46:230-238. * Loc. cit. s Loc. cit.
237
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D. C. WARREN AND LESTER KILPATRICK Kansas Agricultural Experiment Station The observations of various workers have fairly definitely established the fact t h a t the m a x i m u m functional life of the sperm in t h e oviduct of the hen is about one month. D u n n 2 obtained a fertile egg t h i r t y days after the removal of the male a n d Crew 3 recorded the last fertile egg on t h e thirty-second day. The different workers a r e i n agreement t h a t after about 12 or 14 days following t h e removal of the males, a flock will show a r a p i d decline in t h e percentage of fertility. This would indicate t h a t the average span of life of the sperm i n t h e oviduct is reached soon after this period. A n o t h e r explanation would be t h a t the s u p p l y of sperm h a s been depleted t h r o u g h polyspermy or a clearing of t h e oviduct due to repeated passage of eggs. T h a t the latter is a n i m p o r t a n t factor seems improbable since Crew 4 carried out a n experiment utilizing pullets which did not come into production u n t i l two to four weeks following the removal of t h e male. I n this case where the sperm supply had scarcely been d r a w n upon, t h e d u r a t i o n of fertility was not different from t h a t where the females were laying a t t h e beginning of t h e experiment.
POULTRY
SCIENCE
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FlGUKE 1.
A. A freshly released living sperm. B. A fixed and stained freshly released sperm. C. Sperm recovered from the oviduct 17 hours after coition. D. Sperm recovered 4 days after coition. E. Sperm recovered 18 days after coition. The fresh stained sperm show a sharp demarcation at the point of attachment of the flagellum while in the living material there is a gradual tapering in this region. The less pointed ends of the sperm showing no flagella are probably the ones which originally carried the propelling organ. There is little change with age other than the loss of the flagellum.
FERTILIZATION
239
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original male functioned was greatly reduced, indicating that freshness was a factor in their competitive action. His conclusions were based upon a single exchange of males and where only a few females were involved. The writers carried out an experiment of repeated alternations, to determine whether more extensive data would substantiate the findings of Crew. Table 1 shows the results of an experiment covering a period of 134 days in which a Single Comb White Leghorn and a Black Minorca male were alternated five different times, each period of service being 21 days. At the beginning of the last period, the serving male was removed without replacement, in order to compare the duration of fertility without competition with the previous periods where it existed. The eleven hens used were Jersey Black Giants and Black Minorcas. From this group of females the Black Minorca male produced black chicks and the White Leghorn male carrying the dominant white, gave white chicks. Thus the color of the chick would indicate which male's sperm were functioning during the period following each exchange. The vertical lines in table 1 indicate the date of exchange, the exchange being made during the night of the date preceding the line. The experiment covered the period February 8 to June 21. In the table the figure 0 indicates an infertile egg. The interrogation mark is for cases where the embryo died at too early a stage in development for color classification. The letter W is for white chicks and the letter B for black ones. The blank spaces are for days when no eggs were laid. All hens were trapnested and eggs were marked with the hen's number and the date laid. The eggs were placed in the incubator at the end of each week. On the tenth day of incubation all infertile eggs and those containing dead embryos were removed from the incubator. The remaining eggs were broken on the eighteenth day and records made of color, sex, and date laid. Table 1 gives the results of the 1927 season. The results for the 1926 season are found in table 2. This experiment was conducted in the same manner as that for 1927 except that each male served a period of 20 instead of 21 days and the alternation was between a Single Comb White Leghorn and a Jersey Black Giant male. The females in 1926 were all Jersey Black
w h i t e male
0
?BB B
OBB OEB
??? • T \V??B BB B BBB BE BB '".'TT: "TT7T' H W V.T TO F,','B BBBB
PBBB BB
BBBBBB
,B
B
B
B
The sequence of offspring resulting from the males upon a pen of black females. The vertical 0 is for infertile eggs, ? for embryos which died for black ones. Each blank space indicates a day ference to exchange of males.
1
0
3 B
0 E B ?B
BB
B BBB
BB
B B 0?
V? BBB BBBB
OB B OB
0 7?.' 1"W?EBB BB
0
0 00
?0 BO
0
?
00 00
00
0
000 0
00 00
BO 00
BB B ?B ? 3 ?B B 00 B B B
? 0 ? BB? OB BOOOO 0 0 0 0 -
0000 0
? PBB OBO 0 0 0 0
BB BRPBB BBOB
BB
00
0
alternation of a Single Comb White Leghorn and Black Minorca lines in the table indicate the periods of exchange. The figure too early fo rdetermination of color, W for white chicks and B and the position of the letter shows the time of laying with re-
TABLE
B
B? B ? C O B B
OOOWOO
?? ?
B ?B ?B BBE OB ? BB B
BBBB OEBB B ?B BO? BBOO 0 0 0 r>00 0 ) OBOB BBB BBBOBB BBC 3 ?OBB
00 000 0 0 0 3
B B BO 0 0 0 OB
WBBVZ BBBB BBBB BBBBB 3 B \" WTO? ^.VWW S l f W "WW WBBB? ? ?B?BBB
BB BBB? BB ?B ?B E ? BB
BB 00B
0 ? 3 BB BB B B
W W W
000
0000000
BBB
PB
serving
0 ?V? O-TOO 0 0 0 0 0 0 0 C BO,."V,">' vr?™wmPHVW!!V! ? V ^WYTW TT'BBB ?BB0BBBB 30BO0 0 0 0 0
W n'
BB BB
B ? ??B ? B
Ho m a l e
BOBO 0"
ynf •
P." B ' ?B ffW W VTfW W ? BBBB BBB BB? B BB P*B P7TT FFW 1PW lPWff F¥ PWWVT ?BBV.'B
0 0 ? BBBB BB BBB )BB V/?W
serving
?B BBBEBBB B B B
5492
8296
B l a c k male
0B00000C 0 OBB BBBBBBB? B BBEE ?B
V.i.ny
? ?
0 C
Y.V VW C
11
oooc 5000 00B BBB B? BB BE ? .??B V.W.'-' \"\™
00 CT'I" O'.'.'O ?
serving
BBBB 00'." V'C ? BB BB OBBBB B
0 ? 00 ?B0 00CX>?BB BB BBB? BOBBBBE BBB BBBBBBB
B
White male
BBBOB BI ?B B
serving
1 B B ? ? ? ? BB ™ ?•" v:?.v??••"; W ?'•' ?BBBBBBBB ?BB? B BB'VB "TT
ooo oc ")0 0 0 0 0 0 0 0 OBOB B B
W'C 0 0
0 0 ^ 0 30BBBB -BB7BBBBBB0BPBF ? ?0
Black male
0 0 0 0 ilOBBBB BBBB
serving
5419
5394
1270A
1269A
1268A
1266A
serving
OB BBB BB BE ^B BBBBBB BBBB
Slack male
0 OC D B ?B
White m sewing
126 5A wo
1258A
12 57 A
Band H o .
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ta o
©.
to
a
©
to
FERTILIZATION
241
« Loc. cit.
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Giants. In the 1926 experiment no record was made of the embryos which died too early for color classification. Preferential mating enters into the experiment since in table 1 it is seen that three females never mated with the White Leghorn male, while three others produced only a very few offspring from him. In table 2 there is less evidence of this tendency. Since Dunn 6 found that 21 hours was the minimum period between coition and laying of a fertile egg, it should be expected that following an exchange, no chicks would be obtained from the replacing male until the second day. Several chicks were obtained from the replacing male on the third day after the exchange. Records of the exact hour of laying were not kept but third day eggs must have been laid at least 48 hours after mating. The males were exchanged at night so that mating did not take place until the early morning of the first day. The difference between our results and those of Dunn might be explained by the fact that he was reporting upon virgin females while our work was with individuals which were already fully fertilized. If fertilization normally takes place at the time of the release of the ovum from its follicle, either in the ovary or at the time of entrance of the infundibulum, then sperm at these points would have an advantage over those traversing the oviduct. It is possible that, providing fertilization has not taken place earlier, the egg may be fertilized at a much later stage of its passage down the oviduct. If the only sperm in the oviduct are those which are only part way up, then the period between coition and the laying of the egg would be reduced to a minimum. If the assumption regarding the advantage due to position of the sperm in the oviduct is correct, then the period between coition and the laying of an egg fertilized by a newly introduced male would be shorter in virgin than in non-virgin females. There is considerable variation in the length of the period between coition and the laying of the first egg bearing the embryo fertilized by the new male in a replacement. Much of this variation is probably due to the fact that the male does not immediately mate with some of the females in the pen. Also failure of the hens to produce regularly during the first few days
242
POULTRY
SCIENCE
MORPHOLOGICAL CHANGES OCCURRING I N SPERM W H I L E HELD I N T H E OVIDUCT
In order to determine whether any morphological changes could be seen which might explain the inferiority of stale sperm, it was planned to examine those which had been held in the oviduct for varying periods. For this purpose controlled matings of yearling females, known to be free of functional sperm, were carried out. Single Comb White Leghorn hens were used and the male was of the Barred Plymouth Rock breed. Several males were tried out and this particular one was selected because his disposition was such that matings could be obtained whenever desired. At intervals the fertilized females were killed and sperm samples were taken from various regions of the oviduct. The oviduct was removed from the body cavity and slit its entire length. The regions from which the samples were taken were slightly moistened with normal salt solution and the mucus from the inside of the oviduct was lifted by means of a quarterinch platinum loop and transferred to a microscopic slide. A section of the slide the size of the cover glass used, was flooded
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following the exchange or the early mortality of the embryo were factors contributing to the length of the period. The most interesting result shown by tables 1 and 2 is not the exact period at which the new male's sperm start functioning but that once the substituted male's offspring begin to appear, there is seldom any recurrence of the previous male's progeny during the latter's period of service. Frequently, the eggs laid from 3 to 5 days after the first possible coition are fertilized by the new male. The offspring of the previous male seldom appear after the new male's sperm start to function. In all cases where eggs fertilized by the first male are laid after those fertilized by the male succeeding him, it has been a single exception. Since the sperm of the first male function for a much shorter period than appears to be normal from experiments of other workers and as shown by the last section of table 1, it seems that freshness of sperm is a factor where the sperm of two males are allowed to compete. Since this phenomenon occurred in the same manner regardless of the direction of the exchange, it cannot be held to be due to the superiority of the sperm of one male over the other.
FERTILIZATION
243
Whit« nale serving Black male s e r v i n g CGG7 6661
BBOfflO
00
WO
00
0
0 00
0 0
6615
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5451
w w
00 .000 00 00 0
0 0".V
BB B BBBB )BVf W
w
w
71 \'l :txa
>;?:i w w/r.7 v, 7 W 0 0 W Tf W W W 1
C
WO WW
0 0
0
0 0 B 3 B 0
BVT w WTMTW w o w
o t; B a o B
B BB WW W 0 W7TV . TWE-O O B
B B
Vf W W
BB I OB www B ww WIT,*™ O
0 BBS BB > BVWT W W W T.T.' W ff
37; HO W W WWW '.7.717 KW W
l T flW BB BO 03BE jw vr.vo ww YMVWWWW v. T WBBBO B 00 00 0 00 000 0
0
-
B B B nw t m o S B B
v: w
0
viwwc WO W Vffi B B
w WOTWWW
www w w
7003
5403
o
WW
6629
5492
B B B SB C
O . YJWOOW WOW , )0W WO
OW0W0 OBBJ B
White male servijig
ITB vmt oww vnvww w oo cm ww wmvw wwi VWVJW B BBB3B 03BB( B WW; WM7WWW.7 WW V.ViT WWW 0 IV OBB BOBBBB
6618
6628
Black male serving
'W CO 0 OS I B BIf » O
00
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6633 662G
0 YJ .1
White male- serving
0
0 0
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TABLE 2
Results of alternating a Single Comb White Leghorn and Jersey Black Giant males during the 1926 season. The symbols are the same as were used in Table 1.
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with the fluid. The use of the platinum loop for taking the samples is somewhat laborious but this instrument permits thorough flame sterilization between samples. The samples of mucus were taken from the two ends and the middle of the oviduct. The samples were further prepared for studying by drying on a warm radiator and staining with gentian-violet. This stain was used because of its action upon the flagellum as well as other parts of the spermatozoon. After staining, the slides were again dried and mounted in balsam in the usual manner. Slides made from the mid-section of the oviduct were found of little value since the albumin encountered here caused the preparation to be unsatisfactory for study. Material for study was taken from females killed at 6, 17, and 24 hours after mating and at daily intervals until the eighth day. Sperm held in the oviduct for longer periods were collected on the 12th, 15th, 18th and 21st days. Fresh semen was obtained by interposing a watch glass dur-
244
POULTRY SCIENCE
? PAYNE, L. F., 1914. Preliminary report of vitality and activity of sperm cells and artificial insemination of the chicken. Oklahoma Agric. Exper. Sta. Circular 30, 1-8.
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ing copulation. In cases where the above method is not possible the semen may be recovered from the cloaca of the female by use of a spoon. For comparison with sperm recovered from the oviduct the fresh material was prepared and stained in the manner already described. The accompanying drawings show a comparison of freshly ejected sperm, made both from life and fixed material, with those recovered from the oviduct. It will be noted that the only visable change is the loss of the propelling organ. The preparation made six hours after coition showed a number of sperm in all regions of the oviduct. There was practically no change in shape or size of the sperm, but a large number had already lost the flagellum. It is difficult to estimate just what percent still retained this organ since many of the recovered sperm were in positions which made it difficult to determine unquestionably whether or not the flagellum was present. Although the flagellum takes stain rather faintly, this organ could be readily followed to its extremity in mounts of diluted semen. However, in mounts of recovered material the mucus itself took stain to a considerable degree and in many sections of the preparation made the delicate flagellum difficult to trace. The preparation made seventeen hours after coition still showed some sperm with flagella but from a large majority of them were absent. Both at this and the six-hour stage, the flagellum which in fresh sperm is several times the length of the main body, was much shortened. At the,twenty-four hour stage sperm retaining flagella were only rarely found. After this period there were no visable changes in sperm, except a possible shortening and a taking on of a more definite crescent shape. These latter changes may, however, have been due to a difference in the fixation. Payne 7 reports the sperm to be very much reduced in size after the fourteenth day following coition. The writers found considerable variation in size not only in the different preparations but also among the individual sperm on a single slide. The drawings made of eighteenth day material average as large as do the earlier stages. Eighteen days after
FERTILIZATION
245
RESULTS W H E N COMPETING SPERM ARE ALL BEYOND A GIVEN AGE
Since the foregoing studies indicate that the flagellum of the sperm is lost very early, probably in most cases before they are in the oviduct 24 hours then all competing sperm over this age should have equal opportunity. This is assuming that the loss of the tail is the major factor contributing to the handicap of stale sperm. In the previous experiments where two males were alternated upon the same lot of females, the matings were made for a period of three weeks. If an experiment were carried out as before except that within a few days after that exchange, the second male was also removed, then after a 24 hour period the sperm of both males being within the functional age but having lost their flagella, would have equal opportunity. An experiment was outlined to determine if such would be the case. Ten
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coition was the longest period at which recovered sperm could be identified. Since the flagellum probably functions as a propelling structure, its loss soon after the sperm has progressed up into the infundibulum may be the explanation of the observed advantage of fresh sperm over stale ones. Since sperm are capable of fertilization after the loss of the nagellum, it would appear that this is not their only means of locomotion. The worm-like contortions of the head of some of the spermatozoa shown in the prepared material may be taken to indicate that this part of the sperm is sufficiently flexible to allow motion of this sort. Although the flagellum may not be necessary for locomotion, it probably facilitates it so that sperm lacking this structure are not able to compete with those possessing it. It is also of interest to note that in all of the stages studied, the sperm were fully as abundant in the lower as in the upper end of the oviduct. The sperm found in the two ends were compared for the purpose of determining whether those near the vent might not be injured or abnormal ones and because of this, had failed to ascend the oviduct. No evidence was found indicating that the sperm of the vent region differed from those of the upper end of the oviduct. The presence of sperm in all parts of the oviduct at all times may be an indication that the sperm migrate about the oviduct.
246
POULTRY
SCIENCE
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Single Comb White Leghorn females which had previously been with males of their own breed were mated to a Silver Laced Wyandotte male known to be homozygous for rose comb. The pen was made up in the evening and the eggs laid during the next 17 days were incubated. The Wyandotte male was removed from the pen at the end of the third day. Eggs accumulated at the end of each week were placed in the incubator and upon the eighteenth day of incubation the eggs of each lot were broken and recorded for comb type, sex, and date laid. Since the rose comb is dominant to single comb, the offspring of the Wyandotte male had rose combs and the White Leghorn chicks had single combs. A second pen of eight Single Comb Rhode Island Red females previously with males of their own breed were mated to a Light Brahma. This pen was conducted in the same manner as was the other just described except that the feathered and non-feathered condition of the legs was used to distinguish the pure bred Rhode Island Red chicks (unfeathered) and the crossbred (feathered) offspring of the Light Brahma male. Feathering is dominant to non-feathering and the Light Brahma male was known to be homozyous for legfeathering. Table 3 gives the results of these two matings. The twentyfour hundred group are the White Leghorn females and the rest of the numbers are for the Rhode Island Reds. The letter P was used for the pure bred offspring of either breed while the letter X was used for the offspring of either the Silver Laced Wyandotte or the Light Brahma male. The letter 0 is for infertile eggs and the interrogation mark for eggs in which the embryo died too early for classification. As in the other experiments, there are a number of uncontrollable factors which will modify the results here. The results agree with those of the other experiment in that no eggs fertilized by the substituted male were obtained until the third day. The conditions here were slightly different from the previous experiments in that the males were placed in the pens about two hours before night so that opportunity was given for mating during the first evening. In the lot of Rhode Island Red females, the value of much of the data was lost because of the failure of either the Light Brahma or the male which preceeded him to mate with
6H
Female No.
8 44 A 853A 881A 975A 1032A 1037A 1189A 1192A 2403A 2405A 2409A 2419A 2428A 2443A 2455A 2475A 2482A 2453A
1 7
60 p
p
2 P P P X P X P p p
o p p 7
3 P 1
X6 p
0 X p p p 7 P P
4 P X X P X X X X
o o 7
p X X p 0
5 X X p X X 7
P X P P P 0
6 X O X X p 0
o X p
X p
T A B L E 3.
8 P X P X X p p p p 7
O
X p
X 0 0 X X p X
9 P 7 X P X X
10 7 X 0
•x X X 0
X p 0 p p
0
11
o7
12 0
o X
X 0 0
0 p p X p
13 0 0 7
6 0 X
X 0
X
X p
0
p X 0
0
o
0
0 X p
0 p X X X
o
Period of Experiment (days)
7 P X 7 P X 7 X 0 X P P P X
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p
o o
14 0
o o o
0 7 X
O O O
O X
15
16
0 0 0
17
7 X
o
0 0
0 0 0
o o
X 7
o
0 X 0
o
0
0
6
0 7
0
X X X 0
7
6
o
0 0
o
Combined results of two experiments in which females were mated for a period of three days with males of a different breed, following a period of mating with males of their own breed. The first eight females were Single Comb Rhode Island Reds which were mated to a Light Brahma male while the remainder were Single Comb White Leghorns which were mated to a Silver Laced Wyandotte. The period shown is from 1 to 17 days after the starting of the cross-bred mating. The letter P indicates a pure bred offspring, while the letter X is used for cross-bred chicks. The interrogation mark is for cases where the embryo died too early for classification and the figure 0 is for infertile eggs. In several instances there was here (particularly in the case of 2443A) an overlapping of offspring of the two males which was seldom found in Tables 1 and 2.
248
POULTRY
SCIENCE
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some of the females. In the case of female 1032A results were obtained which were not comparable to conditions found in the numerous exchanges made in previous experiments. Here a pure Ehode Island Red chick was obtained eleven days after the second male's sperm had begun to function. More convincing evidence was obtained from the pen of White Leghorn females. A very striking case is that of 2443A where a pure bred offspring was obtained on the first day; a cross bred on the 4th; a pure bred the 5th; a cross bred the 6th; a pure bred the 7th; a cross bred the 9th; three pure breds on the 10th, 11th and 12th; and a cross bred on the 14th. Similar conditions are found in the case of 2455A and 2475A. The records of 2405A, 2419A and 2428A also show exceptional overlapping but not to the degree shown by the first mentioned three. Any overlapping of offspring of exchanged males is exceptional in tables 1 and 2 and in the few instances where it does occur, it is confined to a single individual. There are only seven instances of overlapping shown in tables 1 and 2 and three of them were on the second day, one on the third, two on the fourth and one on the fifth day after the second male's sperm started to function. In some of the matings showns in table 3 it would appear that it is entirely a matter of chance which male's sperm fertilized the egg. The ratio of offspring of the two males would depend upon the relative abundance of the males' sperm. It is clearly demonstrated that following an exchange, the sperm of the first male can function for most of their life span in competition with sperm of the male which succeeds him providing each male's sperm have reached a certain age. Although the cases here cited are not numerous, they are strikingly different from the more numerous exchanges shown in tables 1 and 2. The results obtained from this experiment appear to support the evidence from microscopic study, that loss of the flagellum accounts for the failure of stale sperm to successfully compete with fresh sperm. Both methods of investigation indicate that the casual factor is effective soon after the sperm enters the oviduct. I t should be stated, however, that the evidence is indirect and unobserved changes might be equally as important as those recorded. Freshly ejected sperm were kept in an incubator at 100 de-
FERTILIZATION
249
STALENESS OF SPERM AND EMBRYONIC MORTALITY
Crew8 concluded from his experiments that although sperm were capable of fertilizing eggs after (the sperm) being in the oviduct for 15 to 20 days, "eggs laid after the first week commonly fail to complete their development." Dunn 9 questioned the conclusions of Crew and showed that his own results did not support such a view. The data presented by both Crew and Dunn upon this problem are rather meager. In the experiment presented here, a sufficiently large number of females were used to minimize the effects of individual variability which is always an important factor in problems involving hatchability. In order to reduce the influence of incubator fluctuations, the experiment was run in duplicate series. In the first series 52 females were used and in the second, there were 66. All females were trap-nested and individual hatching records kept. In order to keep conditions uniform the eggs were placed in an electric incubator daily. The trapnests were visited five times daily and the eggs were immediately placed in a small incubator where they were kept until evening, when they were placed in an insulated container and transferred to the s Loc. cit. 9 Loc. cit.
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grees Fahrenheit and examined to determine whether the loss of the flagellum could be observed under these conditions. Motility practically ceased after ten hours and at about twenty hours the sperm began to disintegrate. Both normal salt solution and egg albumin were used to dilute the semen and the material was held under conditions where little evaporation occurred. I t was found that the sperm retained their flagella as long as they lived under these conditions. The results here are not in agreement with the observation made on sperm held in the oviduct since the preparation made six hours after coition showed many of the sperm lacking the flagellum. A possible explanation is that the flagellum is whipped off by the activity of the sperm and conditions provided in the incubator may have retarded the activity of the sperm and as a consequence the flagella were not lost. To the writers this explanation is not entirely satisfactory and the disagreement of results is a fact which remains to be accounted for.
250
POULTRY
SCIENCE
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large incubator in which they were hatched. A recording thermometer was kept in the incubator so that any variations in temperatures would be noted. Exceptionally uniform temperatures were maintained and that all incubation conditions were good is indicated by the hatching percentages which are very good. In both experiments pullets were used which had been isolated from males for several months. Males were placed with the females during the night and the first day's eggs were not used since previous work had shown that they would not be fertile. Beginning with the second day the eggs were handled in the manner already described. After being in the pen eight days the males were removed and the eggs were placed in the incubator daily until practically all of them were infertile. Table 4 gives the daily hatching record for both experiments. Five hundreds forty-one eggs were incubated in the first experiment and six hundred ninety-five in the second. Throughout the second experiment the fertility was somewhat poor and when the egg production decreased soon after the males had been removed, other females which were already in the pen but whose eggs had not been used, were also included. It was on the fourth day after the removal of the males that the eggs of the additional females were first used. There were 22 of these females and since there was no reason why the hatchability of their eggs should differ from those already being included, they were added during the late period when the number of fertile eggs rapidly decreased. The hatchability of the eggs of these females for the first full period (6-9 days) after their inclusion was compared with that of the ones already being included. It was found that the new lot showed an average of 72 percent while the females already being used gave 81 percent hatchability during the same period. The eggs of the new lot of females hatched slightly poorer than did those of the females in the experiment from the beginning, thus it cannot be said that the average hatchability was improved by their inclusion. That the results of the first and second experiment are very similar is additional evidence that the inclusion of the extra females during the later periods did not affect the results of the second experiment. In the first experiment hatching was continued for 14 days
o
TABLE
4.
FIRST EXPERIMENT
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Period Unmated Period 2| 3| 4| 5| 6| 7| 8 1| 2| 3| 4| 5| 6| 7| 8| 9|10|11|12[13]14| 15|16|17| 53|58|55|61|49|54|47 47149141152 j 57146162153155 j 601571531611511 441351361 5125137142140 j 45139 43|44|35|41|46|31|45|37|35|27|23|15|17| 6| 2| 4| 2| 4|15|27|31|30|29|30 34|24|27|26|34|23|33|27|28|21|16|10|11| 4| 2| 1| 1| 80|60|73|74|75|65|77 79 55 77|63 76 74 73|73 80 78 70 67|65|67|100|25|50|
T O T A L OP B O T H E X P E R I M E N T S
Unmated Period 1| 2| 3| 4| 5| 6| 7| 8| 9|10|11|12|13]14| 15|16|17| 19121j16128131126135135132136[34130j391 271 44|35|36| 17118113118124115122123[18111j101 4| 6| 2| 2| 4 | , 2 | 13|10|11|12|18|13|16|18|14|10| 8| 3| 4| 1| 2| 1| 1| 77|56|85!67|79|87|73|78|78 91|80 75|67|50 100|25|50|
SECOND E X P E R I M E N T
Mated Period Unmated Period | 2| 3| 4| 5| 6] 7| 8 1| 2| 3| 4| 5| 6| 7| 8| 9|10|11|12|13|14| 15|16|17| | 28|30|31|34|27|32|24 28|28|25|24|26j20|27|18|23|24|23|23|22|24| 1 1 | 4|18|25|26|23|30|23 26126122123122116123114117j16113111111 [ 4| 1 1 | 3110120121117117117 21|14|16|14|16|10|17| 9|14|11| 8| 7| 7| 3| 1 1 | 75|56|80|81|74|57|74 81|54|73|61|73|63|74|64|82|69|62|64|64|75| 1 1
No. eggs No. fertiles No. chicks Percent hatch
1 | | | |
Mated Period 1 2| 3j 4| 5| 6| 7| 8 | 25|28|24|27|22|22|23 | 1| 7| 12116) 17115| 16 | 1| 5| 7|10|13|12|13 1100171| 58163177 |S0181
No. eggs No. fertiles No. chicks Percent hatch
Mated No. eggs No. fertile No. chicks P e r cent hatch
1 1 1 1
Total 541 393 272
Total 695 293 216
Total 1236 686 488
The effect of staleness of sperm upon embryonic viability. Giving the daily hatchability from the second to the eighth day after the starting of the mating and from the first to the seventeenth day after the removal of the males.
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T H E DAILY R E S U L T S S H O W N I N TABLE 4 TOTALED INTO 3 TO T H E R E S U L T S OF T H E T W O E X P E R I M E N T S ARE COMBINED.
10-13 days
14-17 days
Period
6-i) days
Unmated l-o days
No. eggs No. fertiles No. chicks Percent h a t c h
Period 6-8 days
Mated
2-5 days
TABLE 5.
5 D A Y PERIODS.
227 109 77 70.6
150 124 89 71.8
246 209 145 69.4
216 148 111 75.0
231 82 58 70.7
166 14 8 57.1
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after the removal of the males and in the second experiment, for 17 days. There is no evidence of any progressive change in the percentage of hatchability. In many instances the daily number of fertile eggs was too small to give significant percentages. This was especially true at the beginning and end of the experiments when the percentage of infertility was high. For this reason the results were summarized for longer periods. In table 5 the hatching records given in table 4 have been totaled into 3 to 5 day periods in order to increase the numbers in the group to a value where the percentage would be more reliable. The first group included the eggs laid on the second to fifth days after the mating was made. (The first day's eggs were not incubated.) This was made a four-day period to be comparable with the same length periods in the late stage of the experiment. The second group are eggs laid during the last three days that the males were kept in the pen. The first five days following the removal of the males comprise the third group. The last twelve days were divided into three four-day periods. I t is known that the sperm fertilizing the first group of eggs had been in the oviduct from a few hours to a maximum of 4 days before fertilization took place. (This is allowing 24 hours for the pasage of the egg down the oviduct.) From the second group we cannot state so definitely the age of the sperm fertilizing the eggs since the males had been with the females for a four-day period previous to its beginning but since inatings were occurring during the time, the fertilizing sperm are prob-
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253
RATE OP PRODUCTION AND ONSET OF INFERTILITY AFTER REMOVAL OF MALE
Another factor which might influence the length of period from the time of removal of the male to the onset of infertility TABLE 6.
T H E EFFECT OF STALENESS OF SPERM UPON SEX-RATIOS.
A
COMPARISON OF THE SEX-RATIOS RESULTING FROM E X P E R I M E N T S I N W H I C H FERTILIZATION TOOK PLACE AT VARIANG PERIODS AFTER MATING.
Mated Length of Period 2nd to 5th day 6th to 8th day
Females 37 46 Unmated
1st to 5th day 6th to 9th day 10th to 13th day 14th to 17th day
Period Males 53 55
Period 89 65 38 8
78 65 32 4
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ably relatively fresh. For the third period eggs, the sperm may have had a wide range of age but it is proably the fresher sperm which function. The eggs of the fourth period are known to have a minimum age of five days; those of the fifth period, a minimum of 9 days and the eggs laid during the last period were fertilized by sperm that are 13 or more days old. From the results shown in table 5, one can find no support for the conclusion of Crew that the staleness of the sperm influence the viability of the embryo. The last three periods in the table are all beyond the stage at which Crew found that sperm were capable of fertilizing hatchable eggs. The hatchability for the fourth and fifth periods is very good, the fourth being slightly better than the first period. The fifth period is a little lower but better than the third where most of the sperm must have been relatively fresh. The final period showed a hatchability percent lower than any other period but the few fertile eggs (fourteen) incubated gives this value no special significance. There is little evidence of an effect of age of sperm upon embryonic mortality and it is at least shown that an egg laid 17 days after the removal of the male will hatch into a normal chick.
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I n t e n s i t y 60 percent and above I n t e n s i t y 59 percent and below
Average period of fertility 12.04 days 10.4 days
It is seen that those with the higher average intensity produced fertile eggs slightly longer than did the group with the lower intensity. If the passage of ova down the oviduct were a factor in reducing the period of fertility by depleting the supply of sperm, the opposite result would be expected and the small difference probably has no special significance. Thus it can be said that the rate at which a female produces probably does not affect the length of the period through which she will produce fertile eggs after the removal of the male. SEX EATIO
In the study of the relation of staleness of sperm to embryonic viability records were made of the sex of the chicks. These data have been summarized to show the relation of sex ratio to sperm freshness. Table 6 shows the sex ratios obtained in the
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would be the rate at which a female produces. Polyspermy is known to occur in the chicken and the passage of the egg down the oviduct might also carry out other sperm. Due to the large number of sperm that are introduced at a single mating, it would seem improbable that this would be an important factor but since the data upon this question were available, they are here presented. The females involved in the foregoing discussion were used for this analysis. In calculating the intensity of production, that portion of the period shown in table 4 from the production of the first fertile egg in the mated period to the production of the first infertile egg in the unmated period was used. The rate is expressed in percent and is the quotient of the number of eggs produced divided by the days in the period. Only those females which produced during the entire period were used: The females considered were divided into two lots, those having an intensity of 60 percent or better and those below that value. There were 23 in the upper group, ranging from 60 to 75 percent and 19 in the lower group ranging from 40 to 59 percent. The following results were obtained:
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255
SUMMABY
1. If a male in a mating is replaced by another, the offspring of the replacing male very soon supplant those of the former and there is practically no over-lapping of the offspring of the two males. 2. The flagellum of the sperm is lost during the first day's existence in the oviduct of the female. This may account for the advantage which fresh sperm have over those which have been in the oviduct a longer period. 3. In the case of timed matings, the later introduced sperm lose their advantage after they also have been in the oviduct for more than a day. 4. The viability of embryos is not influenced by the period of
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various periods of the experiment. The totals of chicks for the periods here do not agree with the numbers in the same periods in table 5 since the totals here also include chicks which died in shell after the eighteenth day. During the first period when the sperm fertilizing the eggs are known to be relatively fresh, there was a preponderence of males while in the later periods when the sperm had been in the oviduct a considerable period there was a slight shortage of this sex. I t seems doubtful that these differences have any significance especially since the last division of the mated period and the first after the removal of the males also differ in a similar manner when the sperm functioning in these two periods did not differ greatly in age. Since the female of this species is the heterogametic sex there is no theoretical basis for the supposition that the condition of the sperm would influence the sex ratio. Since the data were also recorded in a form, that information regarding the relation of position of the egg in the cycle and sex were available, the results are given. Only laying cycles of four or more eggs were considered. The first two and the last two eggs of a cycle were compared for the sex of the chicks which they produced. Forty cycles were included and the sex ratio of the first two eggs of cycle was 42 females to 38 males, while the last two eggs of the cycle gave a ratio of 46 females to 34 males. The difference here is very slight and cannot be said to have any significance.
256
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SCIENCE
retention in the oviduct of the sperm which fertilize the eggs from which they hatch. 5. The period between the removal of the male and onset of infertility is not influenced by the rate of production of the female. 6. The staleness of sperm does not influence the sex ratios. 7. The position of the egg in the cycle does not have any effect upon the sex ratios. Downloaded from http://ps.oxfordjournals.org/ at NERL on June 23, 2015
VOL. VIIT
JUNE-JULY, 1929
FERTILIZATION IN THE DOMESTIC FOWL
No.~5 1
T H E E F F E C T OF F R E S H N E S S U P O N T H E COMPETITIVE E F F I C I E N C Y OF SPERM
Crew 5 called attention to t h e fact t h a t when one male was r e placed by another, the period t h r o u g h which the sperm of t h e i Contribution No. 46 Department of JPoultry Husbandry. Read at the annual meeting of Poultry Science Association, held at Lafayette, Indiana, August 21-24, 1928. 2 DUNN, L. C, 1927. Selective fertilization in fowls. POULTRY SCIENCE
6:201-214. s CREW, P. A. E., 1926. On fertility in the domestic fowl. Proc. R. Soc. Edinburgh 46:230-238. * Loc. cit. s Loc. cit.
237
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D. C. WARREN AND LESTER KILPATRICK Kansas Agricultural Experiment Station The observations of various workers have fairly definitely established the fact t h a t the m a x i m u m functional life of the sperm in t h e oviduct of the hen is about one month. D u n n 2 obtained a fertile egg t h i r t y days after the removal of the male a n d Crew 3 recorded the last fertile egg on t h e thirty-second day. The different workers a r e i n agreement t h a t after about 12 or 14 days following t h e removal of the males, a flock will show a r a p i d decline in t h e percentage of fertility. This would indicate t h a t the average span of life of the sperm i n t h e oviduct is reached soon after this period. A n o t h e r explanation would be t h a t the s u p p l y of sperm h a s been depleted t h r o u g h polyspermy or a clearing of t h e oviduct due to repeated passage of eggs. T h a t the latter is a n i m p o r t a n t factor seems improbable since Crew 4 carried out a n experiment utilizing pullets which did not come into production u n t i l two to four weeks following the removal of t h e male. I n this case where the sperm supply had scarcely been d r a w n upon, t h e d u r a t i o n of fertility was not different from t h a t where the females were laying a t t h e beginning of t h e experiment.
POULTRY
SCIENCE
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FlGUKE 1.
A. A freshly released living sperm. B. A fixed and stained freshly released sperm. C. Sperm recovered from the oviduct 17 hours after coition. D. Sperm recovered 4 days after coition. E. Sperm recovered 18 days after coition. The fresh stained sperm show a sharp demarcation at the point of attachment of the flagellum while in the living material there is a gradual tapering in this region. The less pointed ends of the sperm showing no flagella are probably the ones which originally carried the propelling organ. There is little change with age other than the loss of the flagellum.
FERTILIZATION
239
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original male functioned was greatly reduced, indicating that freshness was a factor in their competitive action. His conclusions were based upon a single exchange of males and where only a few females were involved. The writers carried out an experiment of repeated alternations, to determine whether more extensive data would substantiate the findings of Crew. Table 1 shows the results of an experiment covering a period of 134 days in which a Single Comb White Leghorn and a Black Minorca male were alternated five different times, each period of service being 21 days. At the beginning of the last period, the serving male was removed without replacement, in order to compare the duration of fertility without competition with the previous periods where it existed. The eleven hens used were Jersey Black Giants and Black Minorcas. From this group of females the Black Minorca male produced black chicks and the White Leghorn male carrying the dominant white, gave white chicks. Thus the color of the chick would indicate which male's sperm were functioning during the period following each exchange. The vertical lines in table 1 indicate the date of exchange, the exchange being made during the night of the date preceding the line. The experiment covered the period February 8 to June 21. In the table the figure 0 indicates an infertile egg. The interrogation mark is for cases where the embryo died at too early a stage in development for color classification. The letter W is for white chicks and the letter B for black ones. The blank spaces are for days when no eggs were laid. All hens were trapnested and eggs were marked with the hen's number and the date laid. The eggs were placed in the incubator at the end of each week. On the tenth day of incubation all infertile eggs and those containing dead embryos were removed from the incubator. The remaining eggs were broken on the eighteenth day and records made of color, sex, and date laid. Table 1 gives the results of the 1927 season. The results for the 1926 season are found in table 2. This experiment was conducted in the same manner as that for 1927 except that each male served a period of 20 instead of 21 days and the alternation was between a Single Comb White Leghorn and a Jersey Black Giant male. The females in 1926 were all Jersey Black
w h i t e male
0
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The sequence of offspring resulting from the males upon a pen of black females. The vertical 0 is for infertile eggs, ? for embryos which died for black ones. Each blank space indicates a day ference to exchange of males.
1
0
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alternation of a Single Comb White Leghorn and Black Minorca lines in the table indicate the periods of exchange. The figure too early fo rdetermination of color, W for white chicks and B and the position of the letter shows the time of laying with re-
TABLE
B
B? B ? C O B B
OOOWOO
?? ?
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0 ? 3 BB BB B B
W W W
000
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PB
serving
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BB BB
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0 0 ? BBBB BB BBB )BB V/?W
serving
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5492
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B l a c k male
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V.i.ny
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11
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serving
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B
White male
BBBOB BI ?B B
serving
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ooo oc ")0 0 0 0 0 0 0 0 OBOB B B
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0 0 ^ 0 30BBBB -BB7BBBBBB0BPBF ? ?0
Black male
0 0 0 0 ilOBBBB BBBB
serving
5419
5394
1270A
1269A
1268A
1266A
serving
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0 OC D B ?B
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ta o
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to
a
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FERTILIZATION
241
« Loc. cit.
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Giants. In the 1926 experiment no record was made of the embryos which died too early for color classification. Preferential mating enters into the experiment since in table 1 it is seen that three females never mated with the White Leghorn male, while three others produced only a very few offspring from him. In table 2 there is less evidence of this tendency. Since Dunn 6 found that 21 hours was the minimum period between coition and laying of a fertile egg, it should be expected that following an exchange, no chicks would be obtained from the replacing male until the second day. Several chicks were obtained from the replacing male on the third day after the exchange. Records of the exact hour of laying were not kept but third day eggs must have been laid at least 48 hours after mating. The males were exchanged at night so that mating did not take place until the early morning of the first day. The difference between our results and those of Dunn might be explained by the fact that he was reporting upon virgin females while our work was with individuals which were already fully fertilized. If fertilization normally takes place at the time of the release of the ovum from its follicle, either in the ovary or at the time of entrance of the infundibulum, then sperm at these points would have an advantage over those traversing the oviduct. It is possible that, providing fertilization has not taken place earlier, the egg may be fertilized at a much later stage of its passage down the oviduct. If the only sperm in the oviduct are those which are only part way up, then the period between coition and the laying of the egg would be reduced to a minimum. If the assumption regarding the advantage due to position of the sperm in the oviduct is correct, then the period between coition and the laying of an egg fertilized by a newly introduced male would be shorter in virgin than in non-virgin females. There is considerable variation in the length of the period between coition and the laying of the first egg bearing the embryo fertilized by the new male in a replacement. Much of this variation is probably due to the fact that the male does not immediately mate with some of the females in the pen. Also failure of the hens to produce regularly during the first few days
242
POULTRY
SCIENCE
MORPHOLOGICAL CHANGES OCCURRING I N SPERM W H I L E HELD I N T H E OVIDUCT
In order to determine whether any morphological changes could be seen which might explain the inferiority of stale sperm, it was planned to examine those which had been held in the oviduct for varying periods. For this purpose controlled matings of yearling females, known to be free of functional sperm, were carried out. Single Comb White Leghorn hens were used and the male was of the Barred Plymouth Rock breed. Several males were tried out and this particular one was selected because his disposition was such that matings could be obtained whenever desired. At intervals the fertilized females were killed and sperm samples were taken from various regions of the oviduct. The oviduct was removed from the body cavity and slit its entire length. The regions from which the samples were taken were slightly moistened with normal salt solution and the mucus from the inside of the oviduct was lifted by means of a quarterinch platinum loop and transferred to a microscopic slide. A section of the slide the size of the cover glass used, was flooded
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following the exchange or the early mortality of the embryo were factors contributing to the length of the period. The most interesting result shown by tables 1 and 2 is not the exact period at which the new male's sperm start functioning but that once the substituted male's offspring begin to appear, there is seldom any recurrence of the previous male's progeny during the latter's period of service. Frequently, the eggs laid from 3 to 5 days after the first possible coition are fertilized by the new male. The offspring of the previous male seldom appear after the new male's sperm start to function. In all cases where eggs fertilized by the first male are laid after those fertilized by the male succeeding him, it has been a single exception. Since the sperm of the first male function for a much shorter period than appears to be normal from experiments of other workers and as shown by the last section of table 1, it seems that freshness of sperm is a factor where the sperm of two males are allowed to compete. Since this phenomenon occurred in the same manner regardless of the direction of the exchange, it cannot be held to be due to the superiority of the sperm of one male over the other.
FERTILIZATION
243
Whit« nale serving Black male s e r v i n g CGG7 6661
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00
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0
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TABLE 2
Results of alternating a Single Comb White Leghorn and Jersey Black Giant males during the 1926 season. The symbols are the same as were used in Table 1.
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with the fluid. The use of the platinum loop for taking the samples is somewhat laborious but this instrument permits thorough flame sterilization between samples. The samples of mucus were taken from the two ends and the middle of the oviduct. The samples were further prepared for studying by drying on a warm radiator and staining with gentian-violet. This stain was used because of its action upon the flagellum as well as other parts of the spermatozoon. After staining, the slides were again dried and mounted in balsam in the usual manner. Slides made from the mid-section of the oviduct were found of little value since the albumin encountered here caused the preparation to be unsatisfactory for study. Material for study was taken from females killed at 6, 17, and 24 hours after mating and at daily intervals until the eighth day. Sperm held in the oviduct for longer periods were collected on the 12th, 15th, 18th and 21st days. Fresh semen was obtained by interposing a watch glass dur-
244
POULTRY SCIENCE
? PAYNE, L. F., 1914. Preliminary report of vitality and activity of sperm cells and artificial insemination of the chicken. Oklahoma Agric. Exper. Sta. Circular 30, 1-8.
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ing copulation. In cases where the above method is not possible the semen may be recovered from the cloaca of the female by use of a spoon. For comparison with sperm recovered from the oviduct the fresh material was prepared and stained in the manner already described. The accompanying drawings show a comparison of freshly ejected sperm, made both from life and fixed material, with those recovered from the oviduct. It will be noted that the only visable change is the loss of the propelling organ. The preparation made six hours after coition showed a number of sperm in all regions of the oviduct. There was practically no change in shape or size of the sperm, but a large number had already lost the flagellum. It is difficult to estimate just what percent still retained this organ since many of the recovered sperm were in positions which made it difficult to determine unquestionably whether or not the flagellum was present. Although the flagellum takes stain rather faintly, this organ could be readily followed to its extremity in mounts of diluted semen. However, in mounts of recovered material the mucus itself took stain to a considerable degree and in many sections of the preparation made the delicate flagellum difficult to trace. The preparation made seventeen hours after coition still showed some sperm with flagella but from a large majority of them were absent. Both at this and the six-hour stage, the flagellum which in fresh sperm is several times the length of the main body, was much shortened. At the,twenty-four hour stage sperm retaining flagella were only rarely found. After this period there were no visable changes in sperm, except a possible shortening and a taking on of a more definite crescent shape. These latter changes may, however, have been due to a difference in the fixation. Payne 7 reports the sperm to be very much reduced in size after the fourteenth day following coition. The writers found considerable variation in size not only in the different preparations but also among the individual sperm on a single slide. The drawings made of eighteenth day material average as large as do the earlier stages. Eighteen days after
FERTILIZATION
245
RESULTS W H E N COMPETING SPERM ARE ALL BEYOND A GIVEN AGE
Since the foregoing studies indicate that the flagellum of the sperm is lost very early, probably in most cases before they are in the oviduct 24 hours then all competing sperm over this age should have equal opportunity. This is assuming that the loss of the tail is the major factor contributing to the handicap of stale sperm. In the previous experiments where two males were alternated upon the same lot of females, the matings were made for a period of three weeks. If an experiment were carried out as before except that within a few days after that exchange, the second male was also removed, then after a 24 hour period the sperm of both males being within the functional age but having lost their flagella, would have equal opportunity. An experiment was outlined to determine if such would be the case. Ten
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coition was the longest period at which recovered sperm could be identified. Since the flagellum probably functions as a propelling structure, its loss soon after the sperm has progressed up into the infundibulum may be the explanation of the observed advantage of fresh sperm over stale ones. Since sperm are capable of fertilization after the loss of the nagellum, it would appear that this is not their only means of locomotion. The worm-like contortions of the head of some of the spermatozoa shown in the prepared material may be taken to indicate that this part of the sperm is sufficiently flexible to allow motion of this sort. Although the flagellum may not be necessary for locomotion, it probably facilitates it so that sperm lacking this structure are not able to compete with those possessing it. It is also of interest to note that in all of the stages studied, the sperm were fully as abundant in the lower as in the upper end of the oviduct. The sperm found in the two ends were compared for the purpose of determining whether those near the vent might not be injured or abnormal ones and because of this, had failed to ascend the oviduct. No evidence was found indicating that the sperm of the vent region differed from those of the upper end of the oviduct. The presence of sperm in all parts of the oviduct at all times may be an indication that the sperm migrate about the oviduct.
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Single Comb White Leghorn females which had previously been with males of their own breed were mated to a Silver Laced Wyandotte male known to be homozygous for rose comb. The pen was made up in the evening and the eggs laid during the next 17 days were incubated. The Wyandotte male was removed from the pen at the end of the third day. Eggs accumulated at the end of each week were placed in the incubator and upon the eighteenth day of incubation the eggs of each lot were broken and recorded for comb type, sex, and date laid. Since the rose comb is dominant to single comb, the offspring of the Wyandotte male had rose combs and the White Leghorn chicks had single combs. A second pen of eight Single Comb Rhode Island Red females previously with males of their own breed were mated to a Light Brahma. This pen was conducted in the same manner as was the other just described except that the feathered and non-feathered condition of the legs was used to distinguish the pure bred Rhode Island Red chicks (unfeathered) and the crossbred (feathered) offspring of the Light Brahma male. Feathering is dominant to non-feathering and the Light Brahma male was known to be homozyous for legfeathering. Table 3 gives the results of these two matings. The twentyfour hundred group are the White Leghorn females and the rest of the numbers are for the Rhode Island Reds. The letter P was used for the pure bred offspring of either breed while the letter X was used for the offspring of either the Silver Laced Wyandotte or the Light Brahma male. The letter 0 is for infertile eggs and the interrogation mark for eggs in which the embryo died too early for classification. As in the other experiments, there are a number of uncontrollable factors which will modify the results here. The results agree with those of the other experiment in that no eggs fertilized by the substituted male were obtained until the third day. The conditions here were slightly different from the previous experiments in that the males were placed in the pens about two hours before night so that opportunity was given for mating during the first evening. In the lot of Rhode Island Red females, the value of much of the data was lost because of the failure of either the Light Brahma or the male which preceeded him to mate with
6H
Female No.
8 44 A 853A 881A 975A 1032A 1037A 1189A 1192A 2403A 2405A 2409A 2419A 2428A 2443A 2455A 2475A 2482A 2453A
1 7
60 p
p
2 P P P X P X P p p
o p p 7
3 P 1
X6 p
0 X p p p 7 P P
4 P X X P X X X X
o o 7
p X X p 0
5 X X p X X 7
P X P P P 0
6 X O X X p 0
o X p
X p
T A B L E 3.
8 P X P X X p p p p 7
O
X p
X 0 0 X X p X
9 P 7 X P X X
10 7 X 0
•x X X 0
X p 0 p p
0
11
o7
12 0
o X
X 0 0
0 p p X p
13 0 0 7
6 0 X
X 0
X
X p
0
p X 0
0
o
0
0 X p
0 p X X X
o
Period of Experiment (days)
7 P X 7 P X 7 X 0 X P P P X
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p
o o
14 0
o o o
0 7 X
O O O
O X
15
16
0 0 0
17
7 X
o
0 0
0 0 0
o o
X 7
o
0 X 0
o
0
0
6
0 7
0
X X X 0
7
6
o
0 0
o
Combined results of two experiments in which females were mated for a period of three days with males of a different breed, following a period of mating with males of their own breed. The first eight females were Single Comb Rhode Island Reds which were mated to a Light Brahma male while the remainder were Single Comb White Leghorns which were mated to a Silver Laced Wyandotte. The period shown is from 1 to 17 days after the starting of the cross-bred mating. The letter P indicates a pure bred offspring, while the letter X is used for cross-bred chicks. The interrogation mark is for cases where the embryo died too early for classification and the figure 0 is for infertile eggs. In several instances there was here (particularly in the case of 2443A) an overlapping of offspring of the two males which was seldom found in Tables 1 and 2.
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some of the females. In the case of female 1032A results were obtained which were not comparable to conditions found in the numerous exchanges made in previous experiments. Here a pure Ehode Island Red chick was obtained eleven days after the second male's sperm had begun to function. More convincing evidence was obtained from the pen of White Leghorn females. A very striking case is that of 2443A where a pure bred offspring was obtained on the first day; a cross bred on the 4th; a pure bred the 5th; a cross bred the 6th; a pure bred the 7th; a cross bred the 9th; three pure breds on the 10th, 11th and 12th; and a cross bred on the 14th. Similar conditions are found in the case of 2455A and 2475A. The records of 2405A, 2419A and 2428A also show exceptional overlapping but not to the degree shown by the first mentioned three. Any overlapping of offspring of exchanged males is exceptional in tables 1 and 2 and in the few instances where it does occur, it is confined to a single individual. There are only seven instances of overlapping shown in tables 1 and 2 and three of them were on the second day, one on the third, two on the fourth and one on the fifth day after the second male's sperm started to function. In some of the matings showns in table 3 it would appear that it is entirely a matter of chance which male's sperm fertilized the egg. The ratio of offspring of the two males would depend upon the relative abundance of the males' sperm. It is clearly demonstrated that following an exchange, the sperm of the first male can function for most of their life span in competition with sperm of the male which succeeds him providing each male's sperm have reached a certain age. Although the cases here cited are not numerous, they are strikingly different from the more numerous exchanges shown in tables 1 and 2. The results obtained from this experiment appear to support the evidence from microscopic study, that loss of the flagellum accounts for the failure of stale sperm to successfully compete with fresh sperm. Both methods of investigation indicate that the casual factor is effective soon after the sperm enters the oviduct. I t should be stated, however, that the evidence is indirect and unobserved changes might be equally as important as those recorded. Freshly ejected sperm were kept in an incubator at 100 de-
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249
STALENESS OF SPERM AND EMBRYONIC MORTALITY
Crew8 concluded from his experiments that although sperm were capable of fertilizing eggs after (the sperm) being in the oviduct for 15 to 20 days, "eggs laid after the first week commonly fail to complete their development." Dunn 9 questioned the conclusions of Crew and showed that his own results did not support such a view. The data presented by both Crew and Dunn upon this problem are rather meager. In the experiment presented here, a sufficiently large number of females were used to minimize the effects of individual variability which is always an important factor in problems involving hatchability. In order to reduce the influence of incubator fluctuations, the experiment was run in duplicate series. In the first series 52 females were used and in the second, there were 66. All females were trap-nested and individual hatching records kept. In order to keep conditions uniform the eggs were placed in an electric incubator daily. The trapnests were visited five times daily and the eggs were immediately placed in a small incubator where they were kept until evening, when they were placed in an insulated container and transferred to the s Loc. cit. 9 Loc. cit.
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grees Fahrenheit and examined to determine whether the loss of the flagellum could be observed under these conditions. Motility practically ceased after ten hours and at about twenty hours the sperm began to disintegrate. Both normal salt solution and egg albumin were used to dilute the semen and the material was held under conditions where little evaporation occurred. I t was found that the sperm retained their flagella as long as they lived under these conditions. The results here are not in agreement with the observation made on sperm held in the oviduct since the preparation made six hours after coition showed many of the sperm lacking the flagellum. A possible explanation is that the flagellum is whipped off by the activity of the sperm and conditions provided in the incubator may have retarded the activity of the sperm and as a consequence the flagella were not lost. To the writers this explanation is not entirely satisfactory and the disagreement of results is a fact which remains to be accounted for.
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large incubator in which they were hatched. A recording thermometer was kept in the incubator so that any variations in temperatures would be noted. Exceptionally uniform temperatures were maintained and that all incubation conditions were good is indicated by the hatching percentages which are very good. In both experiments pullets were used which had been isolated from males for several months. Males were placed with the females during the night and the first day's eggs were not used since previous work had shown that they would not be fertile. Beginning with the second day the eggs were handled in the manner already described. After being in the pen eight days the males were removed and the eggs were placed in the incubator daily until practically all of them were infertile. Table 4 gives the daily hatching record for both experiments. Five hundreds forty-one eggs were incubated in the first experiment and six hundred ninety-five in the second. Throughout the second experiment the fertility was somewhat poor and when the egg production decreased soon after the males had been removed, other females which were already in the pen but whose eggs had not been used, were also included. It was on the fourth day after the removal of the males that the eggs of the additional females were first used. There were 22 of these females and since there was no reason why the hatchability of their eggs should differ from those already being included, they were added during the late period when the number of fertile eggs rapidly decreased. The hatchability of the eggs of these females for the first full period (6-9 days) after their inclusion was compared with that of the ones already being included. It was found that the new lot showed an average of 72 percent while the females already being used gave 81 percent hatchability during the same period. The eggs of the new lot of females hatched slightly poorer than did those of the females in the experiment from the beginning, thus it cannot be said that the average hatchability was improved by their inclusion. That the results of the first and second experiment are very similar is additional evidence that the inclusion of the extra females during the later periods did not affect the results of the second experiment. In the first experiment hatching was continued for 14 days
o
TABLE
4.
FIRST EXPERIMENT
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Period Unmated Period 2| 3| 4| 5| 6| 7| 8 1| 2| 3| 4| 5| 6| 7| 8| 9|10|11|12[13]14| 15|16|17| 53|58|55|61|49|54|47 47149141152 j 57146162153155 j 601571531611511 441351361 5125137142140 j 45139 43|44|35|41|46|31|45|37|35|27|23|15|17| 6| 2| 4| 2| 4|15|27|31|30|29|30 34|24|27|26|34|23|33|27|28|21|16|10|11| 4| 2| 1| 1| 80|60|73|74|75|65|77 79 55 77|63 76 74 73|73 80 78 70 67|65|67|100|25|50|
T O T A L OP B O T H E X P E R I M E N T S
Unmated Period 1| 2| 3| 4| 5| 6| 7| 8| 9|10|11|12|13]14| 15|16|17| 19121j16128131126135135132136[34130j391 271 44|35|36| 17118113118124115122123[18111j101 4| 6| 2| 2| 4 | , 2 | 13|10|11|12|18|13|16|18|14|10| 8| 3| 4| 1| 2| 1| 1| 77|56|85!67|79|87|73|78|78 91|80 75|67|50 100|25|50|
SECOND E X P E R I M E N T
Mated Period Unmated Period | 2| 3| 4| 5| 6] 7| 8 1| 2| 3| 4| 5| 6| 7| 8| 9|10|11|12|13|14| 15|16|17| | 28|30|31|34|27|32|24 28|28|25|24|26j20|27|18|23|24|23|23|22|24| 1 1 | 4|18|25|26|23|30|23 26126122123122116123114117j16113111111 [ 4| 1 1 | 3110120121117117117 21|14|16|14|16|10|17| 9|14|11| 8| 7| 7| 3| 1 1 | 75|56|80|81|74|57|74 81|54|73|61|73|63|74|64|82|69|62|64|64|75| 1 1
No. eggs No. fertiles No. chicks Percent hatch
1 | | | |
Mated Period 1 2| 3j 4| 5| 6| 7| 8 | 25|28|24|27|22|22|23 | 1| 7| 12116) 17115| 16 | 1| 5| 7|10|13|12|13 1100171| 58163177 |S0181
No. eggs No. fertiles No. chicks Percent hatch
Mated No. eggs No. fertile No. chicks P e r cent hatch
1 1 1 1
Total 541 393 272
Total 695 293 216
Total 1236 686 488
The effect of staleness of sperm upon embryonic viability. Giving the daily hatchability from the second to the eighth day after the starting of the mating and from the first to the seventeenth day after the removal of the males.
POULTRY
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T H E DAILY R E S U L T S S H O W N I N TABLE 4 TOTALED INTO 3 TO T H E R E S U L T S OF T H E T W O E X P E R I M E N T S ARE COMBINED.
10-13 days
14-17 days
Period
6-i) days
Unmated l-o days
No. eggs No. fertiles No. chicks Percent h a t c h
Period 6-8 days
Mated
2-5 days
TABLE 5.
5 D A Y PERIODS.
227 109 77 70.6
150 124 89 71.8
246 209 145 69.4
216 148 111 75.0
231 82 58 70.7
166 14 8 57.1
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after the removal of the males and in the second experiment, for 17 days. There is no evidence of any progressive change in the percentage of hatchability. In many instances the daily number of fertile eggs was too small to give significant percentages. This was especially true at the beginning and end of the experiments when the percentage of infertility was high. For this reason the results were summarized for longer periods. In table 5 the hatching records given in table 4 have been totaled into 3 to 5 day periods in order to increase the numbers in the group to a value where the percentage would be more reliable. The first group included the eggs laid on the second to fifth days after the mating was made. (The first day's eggs were not incubated.) This was made a four-day period to be comparable with the same length periods in the late stage of the experiment. The second group are eggs laid during the last three days that the males were kept in the pen. The first five days following the removal of the males comprise the third group. The last twelve days were divided into three four-day periods. I t is known that the sperm fertilizing the first group of eggs had been in the oviduct from a few hours to a maximum of 4 days before fertilization took place. (This is allowing 24 hours for the pasage of the egg down the oviduct.) From the second group we cannot state so definitely the age of the sperm fertilizing the eggs since the males had been with the females for a four-day period previous to its beginning but since inatings were occurring during the time, the fertilizing sperm are prob-
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253
RATE OP PRODUCTION AND ONSET OF INFERTILITY AFTER REMOVAL OF MALE
Another factor which might influence the length of period from the time of removal of the male to the onset of infertility TABLE 6.
T H E EFFECT OF STALENESS OF SPERM UPON SEX-RATIOS.
A
COMPARISON OF THE SEX-RATIOS RESULTING FROM E X P E R I M E N T S I N W H I C H FERTILIZATION TOOK PLACE AT VARIANG PERIODS AFTER MATING.
Mated Length of Period 2nd to 5th day 6th to 8th day
Females 37 46 Unmated
1st to 5th day 6th to 9th day 10th to 13th day 14th to 17th day
Period Males 53 55
Period 89 65 38 8
78 65 32 4
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ably relatively fresh. For the third period eggs, the sperm may have had a wide range of age but it is proably the fresher sperm which function. The eggs of the fourth period are known to have a minimum age of five days; those of the fifth period, a minimum of 9 days and the eggs laid during the last period were fertilized by sperm that are 13 or more days old. From the results shown in table 5, one can find no support for the conclusion of Crew that the staleness of the sperm influence the viability of the embryo. The last three periods in the table are all beyond the stage at which Crew found that sperm were capable of fertilizing hatchable eggs. The hatchability for the fourth and fifth periods is very good, the fourth being slightly better than the first period. The fifth period is a little lower but better than the third where most of the sperm must have been relatively fresh. The final period showed a hatchability percent lower than any other period but the few fertile eggs (fourteen) incubated gives this value no special significance. There is little evidence of an effect of age of sperm upon embryonic mortality and it is at least shown that an egg laid 17 days after the removal of the male will hatch into a normal chick.
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I n t e n s i t y 60 percent and above I n t e n s i t y 59 percent and below
Average period of fertility 12.04 days 10.4 days
It is seen that those with the higher average intensity produced fertile eggs slightly longer than did the group with the lower intensity. If the passage of ova down the oviduct were a factor in reducing the period of fertility by depleting the supply of sperm, the opposite result would be expected and the small difference probably has no special significance. Thus it can be said that the rate at which a female produces probably does not affect the length of the period through which she will produce fertile eggs after the removal of the male. SEX EATIO
In the study of the relation of staleness of sperm to embryonic viability records were made of the sex of the chicks. These data have been summarized to show the relation of sex ratio to sperm freshness. Table 6 shows the sex ratios obtained in the
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would be the rate at which a female produces. Polyspermy is known to occur in the chicken and the passage of the egg down the oviduct might also carry out other sperm. Due to the large number of sperm that are introduced at a single mating, it would seem improbable that this would be an important factor but since the data upon this question were available, they are here presented. The females involved in the foregoing discussion were used for this analysis. In calculating the intensity of production, that portion of the period shown in table 4 from the production of the first fertile egg in the mated period to the production of the first infertile egg in the unmated period was used. The rate is expressed in percent and is the quotient of the number of eggs produced divided by the days in the period. Only those females which produced during the entire period were used: The females considered were divided into two lots, those having an intensity of 60 percent or better and those below that value. There were 23 in the upper group, ranging from 60 to 75 percent and 19 in the lower group ranging from 40 to 59 percent. The following results were obtained:
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255
SUMMABY
1. If a male in a mating is replaced by another, the offspring of the replacing male very soon supplant those of the former and there is practically no over-lapping of the offspring of the two males. 2. The flagellum of the sperm is lost during the first day's existence in the oviduct of the female. This may account for the advantage which fresh sperm have over those which have been in the oviduct a longer period. 3. In the case of timed matings, the later introduced sperm lose their advantage after they also have been in the oviduct for more than a day. 4. The viability of embryos is not influenced by the period of
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various periods of the experiment. The totals of chicks for the periods here do not agree with the numbers in the same periods in table 5 since the totals here also include chicks which died in shell after the eighteenth day. During the first period when the sperm fertilizing the eggs are known to be relatively fresh, there was a preponderence of males while in the later periods when the sperm had been in the oviduct a considerable period there was a slight shortage of this sex. I t seems doubtful that these differences have any significance especially since the last division of the mated period and the first after the removal of the males also differ in a similar manner when the sperm functioning in these two periods did not differ greatly in age. Since the female of this species is the heterogametic sex there is no theoretical basis for the supposition that the condition of the sperm would influence the sex ratio. Since the data were also recorded in a form, that information regarding the relation of position of the egg in the cycle and sex were available, the results are given. Only laying cycles of four or more eggs were considered. The first two and the last two eggs of a cycle were compared for the sex of the chicks which they produced. Forty cycles were included and the sex ratio of the first two eggs of cycle was 42 females to 38 males, while the last two eggs of the cycle gave a ratio of 46 females to 34 males. The difference here is very slight and cannot be said to have any significance.
256
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retention in the oviduct of the sperm which fertilize the eggs from which they hatch. 5. The period between the removal of the male and onset of infertility is not influenced by the rate of production of the female. 6. The staleness of sperm does not influence the sex ratios. 7. The position of the egg in the cycle does not have any effect upon the sex ratios. Downloaded from http://ps.oxfordjournals.org/ at NERL on June 23, 2015