Metabolism of Turkey Semen as Affected by the Environment of Donor Birds1,2,3

Metabolism of Turkey Semen as Affected by the Environment of Donor Birds1,2,3 I. L. KOSIN Department of Poultry Science, State College of Washington, Pullman (Received for publication September 13, 1957)

REPORT from this laboratory (Burrows and Kosin, 1953) showed that fertilizing capacity of turkey spermatozoa used in artificial insemination was adversely affected when donor birds were subjected to a prolonged period of low ambient temperatures. The same study indicated a seasonal trend in semen fertilizability, as well as in the hatchability of fertile eggs: the former improved with the advent of spring weather and early summer, the latter definitely began to decline in the beginning of May. This decline was more pronounced in the eggs whose embryos were sired by males exposed to the seasonal rise of ambient temperature. Concurrently with the 1953 study {loc. cit.), observations were made on aerobic and anaerobic respiration of semen specimens obtained over a period of time from males subjected to a range of ambient temperatures. The main objective was to observe the effect of temperature stress, directed at the donor bird, on semen metabolism. Design of the investigation also permitted observations on the possible existence of seasonal trends in such metabolism. 1

Scientific Paper No. 1595, Washington Agricultural Experiment Station, Pullman. Projects Nos. 803 and 1040. 2 This investigation was supported, in part, by funds provided for Biological and Medical research by the State of Washington Initiative Measure No. 171, and by federal funds for regional research under the Hatch Amended Act, and conducted at Washington Agricultural Experiment Station. 3 The technical assistance of Mrs. Charlotte Maxwell and Mr. Milton A. Boyd is acknowledged.

As a working hypothesis, an assumption was made that these shifts in the fertilizability and capacity of the spermatozoa to "sire" a viable embryo might be reflected in the metabolic activity of semen. The use of whole semen was rationalized on two grounds. One, preliminary work showed that the turkey seminal plasma has no endogenous respiratory activity, although after the first 90 minutes a low level of oxygen uptake usually could be detected. This, however, was attributed to bacterial growth, which in avian semen proceeds rapidly. Tosic and Walton (1947) made a similar observation on bull seminal plasma. Two, the primary problem was the fertilizing potential of the whole semen. This implied the functional activity of spermatozoa within a substrate, which contained as much as practicable of the original seminal plasma. The complete separation of spermatozoa from their natural substrate would have produced a totally artificial condition with a more likely concomitant distortion in their metabolic pattern. This distortion would have made it difficult to translate such results into terms meaningful to the original problem. The idea that the rate of metabolism of semen and/or of spermatozoa reflects the latter's fertilizing potential is, of course, not new. Walton and Edwards (1938) tested its validity for the bull semen and reported in the affirmative. A number of subsequent investigators have, in general, corroborated this thesis (human semen: Ross et al., 1941; Westgren, 1946; bull semen: Comstock and Green, 1939;

376

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A

METABOLISM or SEMEN

MATERIALS AND METHODS

General: Before dealing with the laboratory phase of the present study, the environments to which donor males were subjected will be briefly described. Environment 1 was a completely enclosed and well insulated room which was maintained at about 65°F. ±5°. In the initial phase of the study (Part A) an additional

environment, environment 2, was provided in the form of a completely enclosed but un-insulated room. In all other respects, the handling of the birds in environment 2 was similar to that in environment 1. Environment 2 was later eliminated when it became apparent that it was not making any substantial contribution to the experimental design of the study. An outdoor pen was environment 3. In environment 1, the birds received at least five foot-candle power of artificial light for 14 hours per day. In environment 3, the artificial light of the same intensity and duration as in environment 1 augmented the natural light. Springhatched males of the Broad Breasted Bronze variety were placed in these environments when they were about 26 weeks of age. However, as it will be pointed out later, in one phase of the study semen samples were not obtained until the donors were over a year old. Male domestic turkeys become sexually mature at 28-30 weeks of age. The floor space in all cases was adequate, (about 10 sq. feet per bird). Feed and water were provided at all times. Altogether, part A involved 19 independent metabolic "runs" (between June 13 and August 29, 1951), and part B, 60 runs (between November 23, 1951, and August 23, 1952). Two entirely different groups of donor males were used in part A and part B. In part A, the males were about 58 weeks old at the beginning of the observation period (June 13). Because of their age, they could be considered to have passed the peak of their reproductive activity. The males in part B, however, were just coming into sexual maturity when they were placed under treatment and semen samples were first obtained from them. In general, semen specimens were obtained twice a week. Because comparisons were made between environments

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Sorensen, 1942; Comstock el al., 1943; Mercier and Salisbury, 1946; Mann, 1948; Blom, 1948; Corrias, 1951; Rollinson, 1951; Erb et al, 1952, 1955, 1956; Ehlers el al., 1953; cock semen: Shaffner, 1948; Shaffner and Andrews, 1948). Not all of the published evidence has been corroborative. Thus, Ghosh et al. (1949) and Schultze and Mahler (1952) failed to find any parallelism between the fertilizing capacity of semen and its metabolic activity. In fact, the latter investigators reported a negative correlation between sperm respiration and semen fertilizing capacity. Bishop et al. (1954) concluded that most of the variation in the oxygen uptake by bull semen can be accounted for by sperm concentration and abnormal spermatozoa. As far as the author is aware, no information directly bearing on the possible existence of a relationship between semen metabolism and viability of avian embryos conceived by the spermatozoa which form part of that semen has been reported. However, circumstantial evidence pointing in that direction exists. It has been known for some time that in the chicken the in vivo spermatozoal senescence is inversely related to hatchability (Nalbandov and Card, 1943). More recently Hale (1955) corroborated this rinding for the turkey. According to him, the effects of sperm aging in this species do not become apparent as early as in the chicken. However, once initiated, the action is more abrupt.

377

378

I. L. KOSIN

Rate of aerobic metabolism was expressed as Zo2 or microliters of oxygen consumed by 108 spermatozoa per hour (Redenz, 1933). Similarly, the anaerobic metabolism was expressed as Zco2 or microliters of CO2 evolved per 108 spermatozoa per hour. Because of the continuous nature of these reactions, recording their progress as a series of independent readings taken at finite intervals appeared to be inappropriate. The 15-minute reading intervals adopted in this study obviously had no other significance than that of convenience. Undoubtedly, the rate recorded at the end of any such interval had been directly influenced by the rate during the preceding moments which, in turn, influenced the reaction rate of subsequent periods. Moreover, the initial readings were alike on all manometers, regardless of the treatment source of the specimen. Consequently, the basic method for reporting the rate was that of cumulative Z values. Each reading was the sum of the preceding readings plus the current reading. Aerobic respiration: Each of the 19 runs of part A and 69 runs of part B included observations on the endogenous respiration of the semen suspended in Ringer's solution. To 0.1 ml. of the original 1:19 semen-Ringer's suspension was added 2.3 ml. of Ringer's solution. The center well received 0.4 ml. of 10% KOH. A more limited series of studies involved the addition of certain substrates to semenRinger's mixture. For example, in part A the effect of sodium succinate on aerobic respiration of the turkey semen was observed. This was to test the possibility that the succinooxidase system is involved in lowering the level of functional activity of turkey spermatozoa after the exposure of donor males to sub-optimal environmental conditions. The methylene

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and not individuals, all observations were based on pooled semen specimens from all males within environments. No attempt was made to balance an individual male's semen contribution to the pooled specimens on different days. The ejaculates were secured in the morning and delivered to the laboratory within 15 minutes of the last ejaculation. The usual collection period extended over an hour. To avoid bias, the order of handling donor groups was rotated. Once in the laboratory, semen specimens were immediately strained through several thicknesses of sterile gauze to remove urate clumps and other extraneous matter. A sample of each specimen was kept for counting by the usual haemocytometer technique. The remainder were suspended in avian Ringer's solution (after Romanoff, 1943), and buffered at pH 7.3, in the ratio of one part semen and 19 parts of Ringer's. Lardy and Phillips (1943) reported 7.25 to be the optimum pH for endogenous respiration of the cock semen. All metabolic determinations were made using a Warburg type manometer and flasks measuring 12-14 ml. in volume. Each treatment in every run was replicated at least twice. As a standard procedure, each flask received 2.4 ml. of the semen-Ringer's mixture in the main chamber. The temperature of the water bath was maintained at 40°C. No flasks were placed in the water bath until the entire series was ready. Following the initial 10 minute equilibration period, readings were taken at 15 minute intervals until the reaction was considered to have ceased in most, if not all, manometers. In practice, this meant discontinuing runs after 3-5 hours. For purposes of statistical analysis the data were, with few exceptions, limited to the readings taken in the first three hours.

379

METABOLISM OF SEMEN

These metabolites were added in the following amounts: sodium succinate, 0.3 ml., 0.5 M.; cytochrome c, 0.4 ml., 10-* M.; sodium pyruvate, 0.3 ml., 0.5 M.; aluminum chloride, 0.3 ml., 10 -3 M. In each case, the volume of the semenRinger's suspension was adjusted to bring its final volume to 2.4 ml. Unfortunately,

most of the runs involving these metabolites were carried out in July and August, 1952, when environment 3 semen was in short supply. Consequently, some of the tests in this series were limited to environment 1 semen. Anaerobic glycolysis: This phase of the investigation was confined to the 1952 study only. The reaction proceeded in an atmosphere of 95% N2 and 5% CO2 gas mixture which was passed through the manometric system for 5 minutes. Before this the semen-Ringer's suspension had been saturated with this mixture by bubbling it through the suspension for 15 minutes. 0.2 ml. of 0.1 M. glucose was added from the side arm following equilibration. RESULTS

The month by month temperature range to which donor birds in environment 3 were subjected is shown in Table 1. Although all manometric readings were adjusted to sperm count before analysis, treatments were compared on the basis of spermatozoal numbers. Table 2 shows sperm count data. There were no statistically significant differences in sperm counts between treatments when the data were subjected either to analysis of variance or to t test. However, such difTABLE 1.—Monthly fluctuations 0} ambient temperature in environment 3 Temperature, °F. Month

Mean

Year

May 1951 June July August September October November December January 1952 February March April May June July August

Max.

Min.

Monthly

66 71 83 80 74 56 43 33 31 39 44 62 66 70 82 81

42 48 55 54 49 40 32 21 21 27 30 37 45 49 54 55

54 59 69 67 62 48 37 27 25 33 37 50 55 60 68 68

Jtiignest

l^ow est

88 80 96 92 88 83 53 52 43 48 65 84 82 88 98 93

30 33 46 43 39 22 21 4 -8 15 21 25 34 39 40 42

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blue decolorization method of estimating semen fertilizing quality (Beck and Salisbury, 1943) is based on that assumption. MacLeod (1943) reported that the presence of succinate as a substrate greatly increases the respiration rate of human sperm. Kosin (1944) corroborated this finding for the cock semen. Accordingly, some of the 19 runs in part A, in addition to measuring endogenous respiration, also involved the addition of 0.1 ml. of 0.1 M. sodium succinate to the semen-Ringer's suspension. The succinate was added before the equilibration period. In part B the compounds used, in addition to sodium succinate, included sodium pyruvate, cytochrome c, and aluminum chloride. Spermatozoa of many species can metabolize sodium pyruvate (Humphrey, 1950; Humphrey and Mann, 1949; Melrose and Terner, 1953). The inclusion of AICI3 was rationalized on the basis of the work of Horecker et al. (1939), who found that the addition of this compound was necessary for the maximum function of succinic dehydrogenase. This point was further elucidated by Schneider and Potter (1943). There is considerable evidence that cytochrome c is necessary for maximum respiratory activity of various kinds of tissues (Potter and Schneider, 1942). Mann (1951) and Zittle and Zitin (1942) showed that cytochrome oxidase is present in human and bull spermatozoa. White (1954) reported that the addition of cytochrome c to mammalian spermatozoa suspension improves motility.

380

I. L. KOSIN TABLE 3.—Mean Zo2 values at the end of each hour. Data not cumulated. Part A

Environment I Environment 2 Environment 3

Hour

25Environment 1 Environment 2 Environment 3

Zo 2 2 °-l

1

2

3 4 Hour of reoction

5

FIG. 1. Cumulated Zo2 at the end of each hour. Substrate: Ringer's. Part A.

ferences, not unexpectedly, were found to exist among specimens within treatments. The lower mean values for sperm counts obtained in the first set of experiments (Nos. 3-21) and higher variability could be due to (1) population differences (age of donor birds and sampling), and (2) errors of estimate. It will be recalled that a different set of donors was involved in each of the two phases of the present investigation. The cumulative Zo2 values for semen collected from environments 1 and 3 in June-August, 1951 are presented graphically in Figure 1. It will be observed that the environment 3 semen shows a depression of aerobic respiration after the second hour. This depression was accelerated with time. Environment 1 semen showed less depression in Zo2 values than either of the other two groups. The correspond-

2nd

3rd

4th

5th

9.43 10.10 10.00

6.69 6.21 5.88

5.64 5.23 4.61

3.88 3.22 3.68

3.50 3.40 3.15

ing non-cumulated data are summarized in Table 3. The significance of these trends, however, should be accepted with some reservation: difference between three treatments, as tested by an analysis of variance, were not significant at P<0.05 though they were significant at P < 0 . 1 . Comparable observations in part B, which extended over a longer period than those of part A (from November, 1951 to August, 1952), corroborated these earlier results on the comparative standing of treatments 1 and 3 (Figure 2). Again, there was a tendency for the difference between the two groups to be accentuated, this time after the first hour. The overall difference in part B in the respiration rate of environment 1 and environment 3 semen was statistically highly significant (P<0.01). Moreover, there was 25-1 Environment

I

Environment 3

Zo,

15

TABLE 2.—Statistical constants for sperm counts (XiO 4 ) Environment 1 X

3 through 21 775 25 through 78 982

Environment 2

Environment 3

a

X

a

x

a

206 138

698

163

746 1,015

212 191

1

2 Hour of

3 4 reaction

FIG. 2. Cumulated Zn2 at the end of each hour. Substrate: Ringer's. Part B.

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10-

1st

381

METABOLISM OF SEMEN 10 -i Environment

3

8 7 6

xZo 2 5-1 4 3 2 I

Nov. Dec. Jan. Feb. Mar. Apr. May June July Aug.

FIG. 3. Effect of season on mean hourly Zo2. Part B.

a highly significant interaction in reaction rate between hour and treatment. The interaction corroborates the validity of the above observation on the relatively faster hourly decline in oxygen uptake by the environment 3 semen. Beginning with the February runs, the difference between two groups in the rate of decline of endogenous respiration became marked by the end of the first hour. Jn fact, the first decline was considerably more marked than the declines after the second and third hours (Figure 3). Note that as the season progressed through spring and summer, the rate of decline in the first two hours, particularly in the first hour, was progressively accentuated in environment 3 semen. There was only a slight suggestion of this trend in the semen from environment 1 donors. TABLE 4.—Effect

The two groups show much overlapping in the first three months. After this, the rates of oxygen uptake begin to separate, particularly in the latter part of the season. Up to the end of January, the cumulative Zo2 values for the environment 1 semen surpassed those of environment 3 in the ratio of 5:4. From that time on, the corresponding ratio widened to 26:8. The chi-square test indicated the latter ratio to be a significant deviation from chance distribution. Tables 4 and 5 show the effect of adding three metabolites and aluminum chloride to the semen-Ringer's suspension on the rate of oxygen uptake. Because, for operational reasons, these metabolites were not always studied concurrently, it was necessary to use a series of different con-

of sodium succinate on aerobic respiration. Cumulated Zo2 at the end of each hour. Part A Environment 1

Environment 2

Environment 3

Hour

Hour

Hour

Substrate 1 Sodium succinate & 9.2 Ringer's 9.8 Ringer's only

2 16.1 16.9

3

4

1

22.2 23.1

25.9 27.0

8.4 9.8

2 13.8 15.5

3

4

1

19.2 20.8

22.5 23.9

9.6 10.8

2 15.7 16.7

3

4

22.4 22.0

26.9 25.8

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Because of the method of collecting and pooling the semen, each donor group within an environment represented a shifting population. Some males failed to produce ejaculates consistently. As a result, the individual males' contributions to a pooled sample varied from run to run. This fact, together with other unavoidable sources of experimental error (such as pollution of specimens with urates, occasional fecal matter and "spontaneous" agglutination of spermatozoa) produced a pattern of considerable overlapping in Zo2 values, both within and between environments. This overlap is illustrated in Figure 4, which plots these values obtained between November, 1951 and July, 1952.

Environment 1

9

382

I. L. KOSIN

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Dates semen collected

FIG. 4. Seasonal fluctuations of cumulated Zn2. Part B. Arrows in 1952 point out days on which the relative standing of ZQ values for semen from environments 1 and 3 was reversed.

trol Zo2 determinations (listed under "none" in the tables) to make these comparison valid. The Zo2 values in Table 4 for the sodium succinate-augmented substrates appear to be too high, It is suspected that the high readings were caused by a consistent under-estimation of sperm numbers in part A of the TABLE 5.—Effect

study (see Table 2). This possible source of error was believed to have been eliminated in part B. However, even if such an error was involved in part A, it applies to all estimates. Hence the values in Table 4 are comparable with one another, In this study the exogenous sodium succinate failed to raise the rate of oxygen

of different metabolites on aerobic respiration. Cumulated Zo2 at the end of each hour. Part B Environment 1

Environment 3

Hour

Hour

Metabolite added to Ringer's

Sodium succinate Sodium succinate+AlCls None Sodium succinate+cytochrome c Sodium succinate+cytochrome c+AlCl Sodium pyruvate Sodium pyruvate+AlCls None AlClj None Sodium pyruvate+cytochrome c Sodium pyruvate+cytochrome c+AlCls None

1

2

3

4

5

1

2

3

4

5

3.6 4.4 6.7

7.2 7.5 11.9

10.9 11.0 16.2

14.1 13.8 20.1

16.9 16.9 23.1

5.4 6.5

9.2 10.7

12.3 14.4

15.4 17.8

18.9 20.0

4.9

9.3

12.6

16.0

18.7

5.0

10.7

14.6

18.1

20.9

4.5 5.8 8.6 7.3

7.7 12.3 16.2 12.3

10.9 18.3 22.8 15.9

13.6 23.9 28.1 18.9

16.1 28.9 32.4 21.2

5.0

8.0

10.9

13..6

16.1

10.7 6.5

19.5 10.9

26.6 14.4

33 0 17 3

37.7 19.6

5.7 6.7

9.6 11.7

12.5 15.8

14.5 19.5

15.8 22.2

4.9 6.5

8.4 10.7

10.9 14.4

12. 17.

13.2 20.0

10.8

20.3

27.1

34.4

39.5

9.8 6.6

17.8 11.4

24.1 14.9

30.3 15.9

34.2 19.0

10.3

18.9

26.0

32.8

38.1

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2.2

383

METABOLISM OF SEMEN

DISCUSSION The first hour Zo2 values for endogenous respiration obtained in this study closely approximate those reported for the

3.4 3.2

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3

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Log Zco 2 2.6

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Dates semen collected FIG. S. Seasonal fluctuations of cumulated Zco2- Part B.

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lots showed no such differences. The results of anaerobic glycolysis were less clear-cut. Plotting the cumulative Zco2 values for individual experiments revealed no obvious seasonal trends (Figure 5). At the same time, the cumulative Z(x>2 hourly readings for the first three hours, regardless of the season, showed an apparent tendency for the environmental groups to separate following the first hour. This time Zco2 values of the environment 3 semen were higher than those of environment 1 (Figure 6). An analysis of variance of these data failed, however, to demonstrate the existence of statistically significant differences between treatments. Neither was there a sizable hours X treatment interaction (P<0.1). Therefore, the apparently growing differential between environments 1 and 3 in the hourly ZCo2 values during the periods of relatively low and relatively high ambient temperatures, shown in Figure 6, should be considered with reservation.

uptake. In fact, the Zo2 values, as summarized in Table 4 and 5, suggest that addition of this metabolite depressed respiration. The depression, however, was not statistically significant. The addition of either cytochrome c or A1CU, or both, to sodium succinate did not change this picture. In fact, when AICI3 was added to the semen-Ringer's suspension, respiration was markedly depressed (P<0.01). Parenthetically, it should be stated that pH readings were not affected by AICI3. Both environmental groups responded alike in this respect. By contrast, adding sodium pyruvate to the substrate caused a marked rise in respiration rate (Table 5), particularly when augmented by cytochrome c, with or without AICI3. When cytochrome c was included, the rate of oxygen uptake at the end of five hours was raised to almost double that of the control rate. These intra-environment differences were found, by / test, to be highly significant (P<0.01). Similar differences were shown to exist on the interenvironmental level for the substrates containing sodium pyruvate, and AICI3, with or without cytochrome c. The corresponding control

384

I. L. KOSIN Environment 1 Environment 3 3rd. hour

Zco,

^ "

^ ^ ""

-

__— -

-

^

—;j»-—«%-"

^"

2nd. hour

*

1st. hour

_—-"^"'-'""' February

March

April

May

June

chicken by Lardy and Phillips (1943). This result indicates that when appropriate adjustments are made for species differences in sperm concentration, the quantitative aspects of oxidative activity are alike in the two animals. The metabolism of turkey semen specimens was found to reflect, to a degree, the effect of one type of environmental stress i.e., high ambient temperature. Although there is no direct evidence on the point in this study, a postulate is made that functional capacity (from standpoint of fertilizing capacity and "siring" a livable zygote) of such semen is also likely to be depressed. This is based on an earlier finding of Burrows and Kosin (1953) that semen from males subjected to an "outdoor" environment similar to that used in the present study had a lower fertilizing capacity. Also later, another study from this laboratory showed that turkey males, exposed to all the fluctuations of ambient temperature normally experienced in Pullman during both early and late breeding seasons are not so effective as breeders (fertilizability, and siring livable embryos) as are those which have received reasonable protection from such temperature stress (Kosin and Mitchell, 1955, 1 and 2). Sperm concentration and mating rate, of course, may be responsible in part for this depression in natural mating, even

There is evidence (Ely el al, 1942) that at least in some species the rate of oxygen consumption by spermatozoa is correlated with their survival rate in vitro, as measured by motility. In turn, this quality is correlated with their fertilizing capacity (Swanson and Herman 1941, 1944; Bishop et a!., 1954). While no similar observations were made in the present study, it is not inconceivable that the environment 3 spermatozoa, because of its impaired oxidative activity, had a shorter functional life span both in vitro and in the oviduct. Limited observations in this study on semen pH values corroborate an earlier observation (Burrows and Kosin, 1953) that no clear-cut pattern exists in the range of these values between environmental groups. The work of Bogdonoff and Shaffner (1954) shows that within the pH range of 6.0 to 8.0, the fertilizing capacity of undiluted chicken semen remains unaffected by shifts in pH. Therefore, it is concluded that the hydrogen ion concentration of the semen within the limits observed in the study did not affect the functional capacity of turkey semen. This study has shown that the turkey

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FIG. 6. Effect of season on mean hourly Zco2. Part B.

though there is no clear-cut evidence for consistent seasonal trends in the concentration of spermatozoa in turkey semen (Burrows and Kosin, 1953; Law, 1957). At any rate, because the "Z" value corrects for sperm concentration, the latter factor can be eliminated from any further consideration in the analysis of aerobic and anaerobic metabolism data obtained in this study. At least a partial answer to the problem of reduced functional capacity of semen from "exposed" donors appears to lie in the fact that semen (or in essence, spermatozoa) from such males shows an altered metabolic pattern: a depression of oxidative processes and a possible increase of glycolytic activity.

385

METABOLISM OF SEMEN

The failure of the turkey semen to metabolize sodium succinate which was added to the substrate was somewhat surprising, in view of the contrary situation in the chicken (Kosin, 1944). One can not generalize from the fact that succinate is metabolized by semen of several widely separated species (Barron and Goldinger, 1940; MacLeod, 1943) because other evidence shows that in some species semen does not possess this capacity (Humphrey and Mann, 1949; Humphrey, 1950). The inability of turkey semen to respond to an exogenous succinate need not, of course, imply the absence of the succinodehydrogenase system in its metabolic pattern. A more likely explanation is the existence of abundant supply of the endogenous succinate in the turkey seminal fluid, making the enzyme(s) incapable of utilizing the exogenous supply of this metabolite. The same reasoning, with one reservation, can be applied to cytochrome c: if one assumes that the latter penetrated the intact sperm cells. Otherwise, a change in toxicity may have been involved. It appears that in this study the

succinodehydrogenase system was not directly responsible for differences in the respiration rate of semen from two environments. The data in this investigation on pyruvate metabolism are limited. Yet there is an indication that the semen from environments 1 and 3 markedly differed in their ability to respond to sodium pyruvate. In one instance (Table 5) the environment 3 semen consistently metabolized sodium pyruvate and AICI3 at a higher rate than that of environment 1, even though the corresponding control groups in both environments failed to show this difference. Again, as was the case with cytochrome c, an unanswered question is: did AICI3 penetrate into spermatozoa or merely exert its influence through a change in toxicity? Essentially the same results to the above were obtained when cytochrome c was incorporated in the substrate, in addition to sodium pyruvate and A1C13. Unfortunately no "control" semen was available in this case from environment 3 donors. However, the Zo2 values for the sodium pyruvate+AlCU+cytochrome c semen paralleled closely the corresponding readings for the sodium pyruvate+AlCl 3 groups. It appears safe to assume, therefore, that had the control semen been available in environment 3 for the former runs, its Zo2 values would have agreed closely to the corresponding environment 1 Zo2 estimates. SUMMARY

A series of studies was conducted on the oxidative and glycolytic activity of the turkey semen provided by donor birds maintained, in the main, under two sets of environmental conditions. Environment 1 was represented by a completely enclosed, windowless pen in which the air temperature was maintained at

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semen can carry on both active oxidative and glycolytic activity, and thus, both pathways presumably are used in the production of energy. This conclusion agrees with the situation in the chicken (Kosin, 1944) and in the bull (Lardy and Phillips, 1941a), but is contrary to the reported metabolic pattern in a number of mammals (MacLeod, 1943,1950) where glycolysis is the main source of energy. It is possible that when oxidative pathways in the turkey semen are partially blocked by environmental stresses directed at the donor organism, the glycolytic activity acquires additional importance as a source of energy for the spermatozoa. Such rerouting of energy producing activity has been demonstrated for the bull spermatozoa by Lardy and Phillips (1941b).

386

I. L. KOSIN

All observations on semen metabolism were based on pooled semen from each environmental group. As a general rule, 0.1 ml. of semen was added to 2.3 ml. of avian Ringer's buffered at pH 7.3. The results were: (1) No significant differences in sperm counts were observed between treatment groups. (2) In both phases aerobic respiration was depressed in the environment 3 semen. Depression first became apparent after the first hour of metabolic "run." Specimens obtained in summer months (second phase) showed the greatest and most consistent depression as compared to environment 1 semen. (3) Adding either sodium succinate or cytochrome c, or both, had no effect on the endogenous respiratory rate. AICI3 depressed it.

(4) Sodium pyruvate was effective in raising aerobic respiration, even more so when cytochrome c was added to the substrate. The environment 3 semen metabolized sodium pyruvate at a higher rate than semen from environment 1 donors. (5) There were no clear cut differences in anaerobic glycolysis between semen from environments 1 and 3. However, a slightly (statistically non-significant) increase in the rate was observed for the latter group. REFERENCES Barron, E. S. G., and J. M. Goldinger, 1941. Effect of iodoacetate and malonate on respiration of sea-urchin sperm. Proc. Soc. Exper. Biol: Med. 48: 570-574. Beck, G. H., and G. W. Salisbury, 1943. Rapid methods for estimating the quality of bull semen. J. Dairy Sci. 26: 483-494. Bishop, M. W. H., R. C. Campbell, J. L. Hancock and A. Walton, 1954. Semen characteristics and fertility in the bull. J. Agric. Sci. 44: 227-248. Blom, E., 1948. Om spermaunders^gelsemet der hos Tyren. Medlemsbl. danske Dyrlaegeforen. 31: 446-462. Bogdonoff, P. D., and C. S. Shaffner, 1954. The effect of pH on in-vitro survival, metabolic activity, and fertilizing capacity of chicken semen. Poultry Sci. 33: 665-669. Burrows, W. T., and I. L. Kosin, 1953. The effects of ambient temperature on production and fertilizing capacity of turkey spermatozoa. Physiol. Zool. 26: 131-146. Comstock, R. E., W. W. Green, L. M. Winters and A. W. Nordskog, 1943. Studies of semen and semen production. Minnesota Agric. Exper. Sta. Tech. Bull. 162: 1-55. Corrias, A., 1951. Valutazione della capacita fecondatira dei tori mediante la prova della ridurtasi sul materials spermatico. Zootec. e Vet. 6: 4 8 58. Ehlers, M. H., F. H. Flerchinger and R. E. Erb, 1953. Initial levels of fructose and citric acid in bull semen as related to fertility. J. Dairy Sci. 36: 1020-1026. Ely, R. E., H. A. Herman and C. F. Winchester, 1942. Studies of respiration rate of dairy bull

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65°F.+ 5°. By contrast, environment 3 was on outdoor pen in which the males were subjected to all the usual flucutations of atmospheric conditions. Environment 2, used only in the first phase of the study, was an enclosed pen. This pen protected the birds from some of the more violent temperature fluctuations, rain and wind, but did not afford the same temperature uniformity as environment 1. All environments were provided with adequate artificial light for 14 hours per day. The males, of the Broad Breasted Bronze variety, were 26 weeks old when placed in these environments. The study consisted of two phases: from June 13 to August, 1951; and from November 23, 1951 to August 23, 1952. Two entirely different groups of males were used in each of the two phases. In the first phase the males were 58 weeks old when the first semen samples were collected from them; in the second phase they were 30 weeks of age.

METABOLISM OF SEMEN

Law, G. R. J., 1957. Ambient temperature and season: factors affecting semen, testes, thyroids, and anterior pituitary of the male turkey. M.Sc. Thesis. State College of Washington. 73 p. MacLeod, J., 1943. The role of oxygen in the metabolism and motility of human spermatozoa. Amer. J. Physiol. 138: 512-18. MacLeod, J., 1950. The semen of the thoroughbred. Cornell Vet. 40:233-248. Mann, T., 1948. Fructose and fructolysis in semen in relation to fertility. Lancet, 254: 446-448. Mann, T., 1951. Studies on the metabolism of semen. VII. Cytochrome in human spermatozoa. Biochem. J. 48: 386-388. Melrose, D. R., and C. Terner, 1953. The metabolism of pyruvate in bull spermatozoa. Biochem. J. 53: 296-305. Mercier, E., and G. W. Salisbury, 1946. The effect of season on the spermatogenic activity and fertility of dairy bulls used in artificial insemination. Cornell Vet. 36: 301-311. Nalbandov, A., and L. E. Card, 1943. Effects of stale sperm on fertility and hatchability of chicken eggs. Poultry Sci. 22: 218-226. Potter V. R., and W. C. Schnedier, 1942. Studies on the mechanism of hydrogen transport in animal tissues. V. Dilution effects in the succinoxidase system. J. Biol. Chem. 142: 543-455. Redenz, E., 1933. t i t e r den Spaltungstoffwechsel der Saugetierspermatozoen im Zusammenhang mit der Beweglichkeit. Biochem. Zeit. 257: 234241. Rollinson, D. H. L., 1951. Fructose estimation and fructolysis of abnormal semen: results obtained in the field. Vet. Rec. 63: 548-552. Romanoff, A. L., 1943, Differentiation in respiratory activity of isolated embryonic tissues. J. Exper. Zool. 93: 1-26. Ross, V., E. G. Miller, Jr. and R. Kurzrok, 1941. Metabolism of human sperm. Endocrinology, 28: 883-893. Schneider, W. C , and V. R. Potter, 1943. The assay of animal tissues for respiratory enzymes. II. Succinic dehydrogenase and cytochrome oxidase. J. Biol. Chem. 149: 217-227. Schultze, A. B., and D. Mahler, 1952. The effect of sodium arsenite on the respiration of bovine semen and the relation of this response to the fertilizing ability of semen. J. Dairy Sci. 35: 906-909. Shaffner, C. S., 1948. The influence of thyroprotein feeding on semen quality. Poultry Sci. 27: 527-528. Shaffner, C, S., and F. N. Andrews, 1948. The influence of thiouracil on semen quality in the

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spermatozoa. Missouri Agric. Exper. Sta. Res. Bull. 353: 1-24. Erb, R. E., M. H. Ehlers and F. H. Flerchinger, 1952. Modified resazurin reduction test for estimating fertilizing capacity of bull semen. J. Dairy Sci. 35: 881-888. Erb, R. E., F. H. Flerchinger, M. H. Ehlers and L. E. Mikota, 1955. Metabolism of bull semen. IV. Relationships among physical measurements, metabolic activity and fertility. Washington Agric. Exper. Sta. Tech. Bull. 18: 1-30. Erb, R. E., F. H. Flerchinger, M. H. Ehlers and F. X. Gassner, 1956. Metabolism of bull semen, II. Fructolysis relationships with sperm concentration and fertility. J. Dairy Sci. 29: 326-338. Ghosh, D., L. E. Casida and H. A. Lardy, 1949. A study of the metabolic activity of bull semen and spermatozoa in relation to their fertilizing ability. J. Anim. Sci. 8: 265-270. Hale, E. G., 1955. Duration of fertility and hatchability following natural matings in turkeys. Poultry Sci. 34: 228-233. Horecker, B. L., E. Stotz and T. R. Hogness, 1939. The promoting effect of aluminum, chromium and the rare earths in the succinic-dehydrogenasecytochrome system. J. Biol. Chem., 128:251-256. Humphrey, G. F., 1950. The metabolism of oyster spermatoioa. Australian J. Exper. Biol. Med. 28: 1-13. Humphrey, G. F., and T. Mann, 1948. Citric acid in semen. Nature, 161: 352-353. Humphrey, G. F., and T. Mann, 1949. Studies on the metabolism of semen. V. Citric acid in semen. Biochem. J. 44: 97-105. Kosin, I. L., 1944. Some aspects of the biological action of X-rays on cock spermatozoa. Physiol. Zool. 17:289-319. Kosin, I. L., and M. S. Mitchell, 1955. Ambient temperature as a factor in turkey reproduction. 1. The effect of preheating males and females on their subsequent breeding pen performance. Poultry Sci. 34: 484-496. 2. The effect of artificially lowered air temperature on the breeding activity of males in late spring and in summer. Poultry Sci. 34: 499-505. Lardy, H. A., and P. H. Phillips, 1941a. The effect of certain inhibitors and activators on sperm metabolism. J. Biol. Chem. 138:195-202. Lardy, H. A., and P. H. Phillips, 1941b. Phospholipids as a source of energy for motility of bull spermatozoa. Amer. J. Physiol. 134: 542-548. Lardy, H. A., and P. H. Phillips, 1943. Effect of pH and certain electrolytes on the metabolism of ejaculated spermatozoa. Amer. J. Physiol. 138: 741-746.

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I. L. KOSIN 37: 69-76. Walton, A., and J. Edwards, 1938. Criteria of fertility in the bull. I. The exhaustion test. Proc. Amer. Soc. Anim. Prod. 1938: 254-259. Westgren, A., 1946. Metabolism and sterility of human spermatozoa. Acta Physiol. Scand. 12 (supplement 39): 1-80. White, I. G., 1954. The effect of some seminal constituents and related substances on diluted mammalian spermatozoa. Austr. J. Biol. Sci. 7: 379-390. Zittle, C. A., and B. Zitin, 1942. The amount and distribution of cytochrome oxidase in bull spermatozoa. J. Biol. Chem. 144: 99-104.

The Gross and Microscopic Anatomy of the Digestive Tract, Spleen, Kidney, Lungs and Heart of the Turkey THOMAS D . M A L E W I T Z 1 AND M . L O I S CALHOUN 2

East Lansing, Michigan (Received for publication September 14, 1957)

T

HE few observations reported on normal turkey histology include detailed microscopy of the duodenum by Rosenberg (1941); pancreatic histology by Lucas (1951); and the microscopic anatomy of the small intestine, cecum and rectum of turkey poults by Demke (1954). MATERIALS AND

METHODS

Two-month-old Beltsville White turkey poults from the Zeeland Hatchery, Zeeland, Michigan, were used in this study. Specimens were removed immediately after death; those from the digestive tract first in order to minimize autolysis. An1

Assistant Professor, Pharmacognosy and Pharmacology, College of Pharmacy, University of Florida, Gainesville. 2 Professsor and head, Department of Anatomy, Michigan State University, East Lansing.

terior, middle and posterior sections were taken from the esophagus, small intestine and ceca. The specimens were fixed in Bouins solution, dehydrated and infiltrated according to the butyl alcohol-paraffin mush method of Johnston el al. (1943). Sections were stained with Harris hematoxylin and eosin (Malewitz and Smith, 1955), Weigert's, Van Gieson's, and Mayer's mucicarmine stains. RESULTS AND

DISCUSSION

Gross Anatomy The gross stuctures of the turkey digestive tract, including appendages, are essentially the same as those of the chicken described by Grahame and Bradley (1950) and Calhoun (1954). The beak and structures of the oral cavity are illustrated in Figure 1, and are

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fowl. Poultry Sci. 27: 91-102. S^rensen, E., 1942. Metoder til Unders^gelse af Spermas Fertilitet. Maanedsskr. Dyrlaeger, S3: 593-627. Swanson, E. W., and H. A. Herman, 1941. Variations in bull semen and their relation to fertility. J. Dairy Sci. 24: 321-332. Swanson, E. W., and H. A. Herman, 1944. The correlation between some characteristics of dairy bull semen and conception rate. J. Dairy Sci. 27: 297-301. Tosic, J., and A. Walton, 1947. Effect of egg-yolk and its constituents on the respiration and fertilizing capacity of spermatozoa. J. Agric. Sci.