Some Chemical Studies of the Female Reproductive Tract and Seminal Plasma of the Male Turkey and Their Relationship to Fertility1

Some Chemical Studies of the Female Reproductive Tract and Seminal Plasma of the Male Turkey and Their Relationship to Fertility1

1170 L. R. MCDOUGALD AND W. M. REID of Eimeria acervulina in light and heavy infections. Avian Diseases, 14: 166-171. Reid, W. M., and M. R. Raja, 19...

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L. R. MCDOUGALD AND W. M. REID of Eimeria acervulina in light and heavy infections. Avian Diseases, 14: 166-171. Reid, W. M., and M. R. Raja, 1963. Eimeria maxima—pathogenicity and incidence in Georgia broilers. Am. J. Vet. Res. 24: 174-178. Reid, W. M., H. E. Womack and J . Johnson, 1968. Coccidiosis susceptibility in layer flock replacement programs. Poultry Sci. 47: 892-899. Reid, W. M., K. Friedhoff, P. Hilbrich, J. Johnson and S. A. Edgar, 1965. The occurrence of the coccidium species Eimeria mivati in European poultry. Z. f. Parasitenk. 25: 303-308.

Some Chemical Studies of the Female Reproductive Tract and Seminal Plasma of the Male Turkey and Their Relationship to Fertility1 E. F. GRAHAM, M. L. SCHMEHL, K. I. BROWN,2 B. G. CRABO3 AND G. ERTEL Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55101 U.S.A. (Received for publication January 11. 1971)

INTRODUCTION

RTIFICIAL insemination in the tur- key (Meleagris gallopavo) has in recent years become almost universal. However, the successful development of a suitable medium for preservation of spermatozoa in vitro for long term storage has not been reported. An approach to preservation in vitro may evolve around the hypothesis that fertility may be related to seminal chemistry, or more important, to the chemistry of the female reproductive tract. It is well established that spermatozoa, once inseminated, live in the female tract for several days prior to fertilization. Little information has been reported on

A

1 Scientific Journal Series Paper No. 7447, Minnesota Agricultural Experiment Station. 2 Department of Poultry Science, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691. 8 Department of Clinical Biochemistry, Royal Veterinary College, Stockholm, Sweden.

seminal chemistry of the turkey. Brown (1959) reported a seminal plasma concentration of 338 mg. (147 mEq./l.) and 113 mg. (29 mEq./I.) per 100 ml. for sodium and potassium, respectively. Considerable work has been conducted and reported on some of the chemical constituents of fowl seminal plasma. An excellent review has been reported by Lake (1966) which in part includes the following data: (see column one, page 1171). The relationships and content of free amino acids in turkey seminal plasma have not been fully investigated. Graham et al. (1964) and Ahluwalia and Graham (1966) reported the free amino acid content in duplicate pooled turkey seminal plasma and duplicate pooled cock seminal plasma samples. They found a considerable difference between the cock and turkey samples. Lake and Hatton (1968) reported differences in free amino acid composition of chicken and turkey seminal plasma.

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Johnson, J., and W. M. Reid, 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor pen experiments with chickens. Exp. Parasitol. 28:30-36. Kemler, A. G., and W. M. Reid, 1961. Incidence of the coccidial species Eimeria necatrix among Georgia broilers as determined by challenge techniques. Poultry Sci. 40: 61-63. Reid, W. M., 1960. The relationship between coccidiostats and poultry flock immunity in coccidiosis control programs. Poultry Sci. 39: 14311437. Reid, W. M., and J. Johnson, 1970. Pathogenicity

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FERTILITY OF TURKEYS Constituents of cock seminal- plasma1 (mg. per 100 ml. seminal plasma)

Constituent

Range Work Cited

4.2 2.1-9.3 132.0 132-205 6.1 6.1-8.4 50.0 39-188 383.0 270-383 7.5 0.06-1.7 0.032 0.7-0.93 7.7-125

Investigators

2,3,5,6 2,3,5 2,3,5 2,3,5,6 6 11 2 2 7,8 4,9,10

1. Lake (1966) 2. Lake, Butler, McCallum and Maclntyre (1958) 3. Lake and Maclndoe (1959) 4. Lake (1962) 5. Lake and El Jack (1964) 6. Takeda (1959) 7. Wales, Scott and White (1961) 8. Pytasz and Klymiuk (1961) 9. Lorenz (1959) 10. Mann (1964) 11. Schindler, Volcani and Weinstein (1958)

A preliminary study was conducted in which the free seminal amino acid content of 17 samples was correlated with fertilizing capacity. All free amino acids and amino compounds, except threonine, were negatively correlated with fertility (3 significantly). Therefore, a more extensive study of free amino acids and amino compound content of seminal plasma was undertaken. Al-Hakim et al. (1970) reported that frequent semen collections affected the free amino acid content of bovine seminal plasma. Therefore, the effects of frequency of collection and season on free amino acid content were studied. Again little has been reported on chemical constituents of the avaian female reproductive tract. El Jack and Lake (1967) showed differences in sodium, potassium, calcium and magnesium from uterine fluids collected at different stages of oviposition. Some histochemistry for the domestic

MATERIALS AND METHODS

Two experiments were conducted and will be presented separately. Experiment 1: Seminal plasma constituents. Source of semen: Inorganic constituents. Semen was collected from K-M1 Large White male line birds. Six individual semen samples were collected from 22 males at weekly intervals over a six week period. These samples were analyzed for inorganic constituents. Source of semen: Amino acids. Another 65 males were collected individually for amino acid analysis. Between semen collections for analysis, semen was collected for insemination to establish individual bird fertility. Due to small quantities of semen for analysis, split samples were not used for analysis and fertility. For the frequency of collection study, semen from 10 Large White toms was col1

K-M Keithley-McPherrin caster, California.

Company,

Lan-

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Calcium Chloride Magnesium Potassium Sodium Phosphorus (total) Zinc Copper Protein Total reducing substances

Means Lake and El Jack (1966)

chicken has been reported by Gilbert et al. (1968), Fujii et al. (1965) and for the quail, Tamura and Fujii (1966). Gilbert et al. (1968) reported that the sperm host glands contain glycogen and that uterine glands and epithelium contain either no carbohydrates or only muco-polysaccharides. They also reported that the host glands secrete acid phosphatase but not potassium. No reports were found relating to the turkey. Apparently no attempts have been made to intensively study the relationship of seminal plasma or female reproductive tract constituents to fertility. A study was designed in an attempt to determine if a relationship exists between seminal plasma composition and fertility.

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Handling of semen: Semen from individual birds for both inorganic constituents and individual amino acid analyses were collected directly by aspiration into a 1 ml. syringe2 and transferred into a glass ampule2 which was sealed and placed into liquid nitrogen ( —196°C). All ampules were labelled by male and collection date and remained in liquid nitrogen until thawed for analysis. Six individual samples per male were pooled for analysis. After thawing and pooling all individual bird samples, they were centrifuged at 15,000 g for 10 minutes and the supernatant decanted. Semen for pooled samples was mixed immediately after collection, volume was recorded and sperm cell concentrations were determined using a photometric method. The pooled samples were placed into ampules which were sealed and placed into liquid nitrogen ( — 196°C.) until thawed for analysis. Plasma for free amino acid analysis was 2

All syringes and glass ampules had been previously washed with non ionic detergent and rinsed in double distilled water.

prepared chemically by a modification of Hamilton's (1962) sulfosalicylic acid (SSA) method. Due to the high free glutamic acid content in turkey seminal plasma, two preparations of each sample were made. The majority of amino acids were in the proper concentration for analysis when 200 [>1. of plasma, 200 jil. of internal standard (1.25 ixM./ml. of norleucine) and 500 [*1. of 6% SSA were centrifuged at 21,500 g for 1 minute. The supernatant provided enough sample for 2 analyses. The second preparation was 10 \il. of plasma, 200 (il. of internal standard and 25 p.1. of 6% SSA which provided 2 analyses where glutamic acid was in a testable range. Fertility. Each individual male was used to inseminate 10 K-M female line birds. Fertility was assessed by a 10 day candle. Inseminations were made using Yto ml. undiluted semen at one week intervals. Fertility data used was the mean fertility of those eggs produced during the six week period when semen was obtained for chemical analyses. Chemical analyses employed. Chemical analyses were performed on a Technicon Autoanalyzer and Amino Acid Analyzer3 and included inorganic phosphate, chloride, calcium, potassium, sodium, total protein, .total reducing substances, and free amino acids and amino compounds. Inorganic phosphate. Inorganic phosphate determination was based on the method of Fiske and Subbarow (1925) automated by Technicon (N-4b). Chloride. The chloride determination employed was based on the method of Zall et al. (1956) adapted for automation by Skeggs (1965) (Technicon: N-5b). 3 Technicon Instruments Corp., 511 Benedict Ave., Tarryton, N.Y. 10591.

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lected and pooled into one sample on the following consecutive collection schedules: ( 1 ) 7 day intervals for 3 collections; ( 2 ) 4 day intervals for 3 collections; (3) 7 day intervals for 3 collections; and (4) 2 day intervals for 3 collections. All collections were completed within a 60 day period. Semen from 10 Large White toms was collected weekly and pooled. Samples were collected during the breeding season from middle January through May. A total of 20 weekly collections were made to study seasonal effect on free amino acids in turkey seminal plasma. Mean temperature values for the week preceding each collection were also recorded. The toms used in all studies were 32 weeks of age at the beginning of each study.

FERTILITY OF TURKEYS

Calcium. The determination of calcium was based on the method adapted for automation by Kessler and Wolfman (1964) (Technicon: N-3a).

Total protein. A modification of the Biuret technique proposed by Weichselbaum (1946) for protein determination, modified by Stevens (1965), was employed (Technicon: N-14b). Reducing substances. Reducing substances were measured by the method proposed by Hoffman (1937) as automated by Technicon Instrument Corporation (Technicon: N-2b). Free amino acids and amino compounds. Free amino acids and amino compounds were analyzed on a Technicon Amino Acid Analyzer by the method of Hamilton (1962) as modified by Dunkelgod (1968). Experiment 2: Some chemical properties of the female reproductive tract of birds of high and low fertility.

were sacrificed and portions of the uterovaginal tissue, magnum, isthmus, infundibulum and thigh tissues were taken. Blood serum samples were also collected. Immediately after extirpation, the samples were placed in non-ionoxed vessels, placed in liquid nitrogen and stored until analyzed. After the samples were thawed, they were prepared independently for analysis of organic and inorganic materials. Inorganic constituents. Samples were prepared for analysis by the emission spectrograph. The frozen tissue was placed in a weighed crucible and placed in a vacuum desiccator at 85°C. for 24 hours. After drying was completed, the samples were weighed and placed in a muffle furnace at 555°C. for 24 hours. One g. dry tissue was weighed and was dissolved in 5 ml. lithium carbonate solvent (0.5% Li 2 C0 3 —1.5% HC1) for 15 minutes. Where the sample dry weight was less than 1 g., proportionate volumes of solvent were used. After sample preparation, 12 inorganic properties were measured on each sample and converted to a dry weight basis. Organic constituents. Immediately on removal from liquid nitrogen, they were thawed and weighed. Two parts distilled water to one part material (w./v.) was added. The sample and water were placed into liquid nitrogen and maserated with mortar and pestal until powder. The samples were then thawed and centrifuged at 15,000 g for 10 minutes. The supernatant material was used for analytical procedures.

Source of material. Females were selected from K-M female line birds. One high and one low fertility female was selected from each of 14 pens. Both females from each pen had been inseminated with semen from the same torn. Fertility of the female was determined in the middle of the breeding season on the basis of the 4 egg settings and hatches just prior to use in this experiment.

Chemical Analyses Employed Lactic dehydrogenase. Lactic dehydrogenase (LDH) analyses were conducted according to the automated method described by Levy et al. (1965).

Methods of handling material. Females

Acid phosphatase. The acid phosphatase

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Sodium and potassium. Sodium and potassium were determined simultaneously with a two-module flame photometer. The method of Burriel-Marti and Ramirez-Munoz (1957) as modified for automation by Technicon (Technicon N-20b) was employed.

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assay employed was a modification by Powell and Smith (1954) of the King-Armstrong method (Technicon: N-7a).

Glutamic oxalacetic transaminase. Glutamic oxalacetic transaminase (GOT) analyses were based on the procedure of Babson et al. (1962) and automated by Technicon Instruments Corporation (Technicon: N-25a). Cholinesterase. Cholinesterase activity was determined by the automated method described by Winter (1960). Statistical Analyses Experiment 1. Means and standard deviation were calculated and the means correlated (Steel and Torrie, 1960) to fertility and percent hatch of total eggs set. The frequency of collection study results were subjected to analysis of variance and Duncan's new multiple range tests. Weekly collection samples were subjected to linear, cubic and quadratic regression analyses. Fertility and percent hatch of fertile eggs were compared with free seminal amino acids and amino compounds using linear correlation analysis. Free seminal amino acid and amino compound content for toms with fertility data and toms without fertility data were subjected to analysis of variance (Steel and Torrie, 1960). Experiment 2. All calculations for organic constituents were based on activity units of enzyme per ml. of supernatant fluid extracted from 1 g. tissue or in 1 ml. blood serum and inorganic constituents on 1 g. dry weight. The chemical composition of high-low fertility groups by area and be-

RESULTS AND DISCUSSION Experiment 1: Seminal Plasma Chemistry. The means and standard deviations of fertility and percent hatch of eggs set of the eggs produced by hens inseminated with semen from 22 toms are shown in Table 1. The seminal plasma composition of these toms is also shown. The results obtained were quite similar to results shown for fowl seminal plasma. Inorganic phosphate content in the seminal plasma of the turkey was lower than that reported by Lake (1962) for total phosphorus in the fowl but higher than he reported for apparent inorganic phosphorus. The chloride content of turkey seminal plasma appeared to be lower than that of the chickens. The potassium content obtained for turkey seminal plasma was somewhat higher than has been reported for fowl seminal plasma but lower than was reported for turkey seminal plasma (Brown, 1959). The sodium content of 166 mEq./l. (383 mg./lOO ml.) was about the same as that reported by Brown (1959) for turkey seminal plasma and for fowl seminal plasma (Lake and El Jack, 1966). Reducing substances in the seminal plasma of the turkey were low in compariTABLE 1.—Some chemical properties of turkey seminal plasma for 22 males of known fertility

% Fertility % Hatch of Total eggs set Inorganic Phosphate mg./lOO ml. Chloride mEq./l. Calcium mg./lOO ml. Potassium mEq./l. Sodium mEq./l. Protein g./lOO ml. Reducing substances mg./lOO ml.

Mean

S.D.

81.0 41.8 4.1 30.7 4.4 16.8 166.6 2.7 33.4

11.8 7. S 0.74 3.25 0.33 1.49 8.4 0.64 5.8

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Alkaline phosphatase. The acid phosphatase assay employed was a modification by Marsh et al. (1959) of the King-Armstrong method (Technicon: N-6a).

tween areas were analyzed and the data were subjected to analysis of variance and Duncan's new multiple range test (Steel and Torrie, 1960).

FERTILITY OF TURKEYS

TABLE 2.—Correlation between percentage fertility and percentage hatch of total eggs set on components in the turkey seminal plasma

% Hatch Inorganic Phosphate Chloride Calcium Potassium Sodium Glucose Protein N=22 *(P<.05) 0.413 ** (P<.01) 0.537

% Fertility

% Hatch

0.913** 0.374 -0.716** -0.452* 0.270 -0.605** -0.272 -0.154

0.505* -0.673** -0.404 0.204 -0.691** -0.060 0.012

TABLE 3.—Means and standard deviations of free amino acids and amino compounds in 135 turkey seminal plasma samples

Amino acid

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

StanMeans in dard StanjuM./ml. Devi- dard ation Error

33.34 14.39 1.24 Total amino acids1 .36 .18 .03 Methionine sulfoxide 5.13 1.38 .12 Aspartic acid 2.50 1.34 .12 Threonine 6.59 2.51 .22 Serine 80.77 21.81 1.88 Glutamic acid 1.40 .60 .05 a-Amino adipic acid 2.51 1.03 .09 Glycine 3.03 1.53 .13 Alanine 1.45 .95 .08 Valine .50 .39 .03 Methionine .78 .56 .05 Isoleucine 1.94 1.28 .11 Leucine .95 .76 .07 Tyrosine .6 .45 .04 Phenylalanine 5.66 1.57 .14 Ammonia 1.13 .97 .08 Ethanolamine .61 .67 .06 Ornithine 1.21 .88 .08 Lysine .78 .35 .03 Histidine 3.39 3.10 .27 Arginine Total amino acids (No. 3-15, 18-21) 114 .11 Total amino compounds (No. 2 -21) 121 .26

1 Excluding glutamic acid and amino compounds (No. 2, 16 & 17).

tion of mammalian sperm with saline is excessive, it leads to permanent loss of motility, metabolic activity and fertilizing capacity. As early as 1934, Milovanov used NaCl as a resistance test. This test indicated that the greater the resistance of spermatozoa to NaCl, the greater the fertilizing capacity. An excess of calcium in seminal plasma that is associated with lowered fertility may be the result of its well-known inhibiting effect on acid phosphatase (Tagnon and Steens-Lievens, 1960; and Steens-Lievens and Tagnon, 1962). Also, calcium ions can depress motility and metabolism of spermatozoa as reported by Mann (1964). Free Amino Acids and Amino Compounds. The free amino acids and amino compounds identified and quantitated are listed in Table 3. An average of 114.11 u,M./ml.

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son to seminal plasma of mammals but fall within the range given for the fowl. Total protein (2.7 grams per 100 ml.) in seminal plasma was considerably higher than the range (0.7 + 0.93) given for fowl plasma but much lower than that for mammals. The correlations between some chemical constituents of seminal plasma and percentage of fertile eggs and hatch of total eggs set are shown in Table 2. Due to the close relationship of percentage of fertile eggs and hatch, similar correlation values were obtained. Significant negative correlations (P < .01) were shown between concentrations of sodium and chloride in the seminal plasma and fertility. A significant negative relationship (P < .05) was shown between the calcium content of seminal plasma and the fertilizing capacity of the spermatozoa. Lake (1960), Wilcox and Wilson (1961) and El Zayat and van Tienhoven (1961) have shown experimental evidence that suggests that chloride can be harmful to the survival of fowl spermatozoa stored in vitro. According to Milovanov (1934) as reported by Mann (1964) potassium, magnesium, phosphate and sulfate are recommended for inclusion into mammalian semen diluents to prevent the swelling of spermatozoa and to protect the "lipid capsule" from the action of NaCl. Mann (1964) also stated that if the dilu-

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TABLE 4.—Mean values of turkey seminal plasma constituents of 12 pooled samples from 10 birds collected at various intervals

l

fJ

2nd 7 day interval

2 day interval

level of significance

2.03 2 . 2 3 17.33 18.33

1.87 14.67

NS NS

* day

•5-'-1.67 12.00 7.55

8.54

8.24

7.74

2 5 . 1 3 b 2 6 . 3 9 b 2 7 . 1 9 b 30.75 a .25 .25 .24 .57 5.98 5.65 5 . 7 8 3.88 1.54 bb 1.68 bb 1.96 ab' b' 2 . 2 3aa 6.77 6.81 6.84 8.48 86.50 77.37 92.67 102.17 a 1.41 bb 1.56 bb 1 . 6 1 bb 2.82 a 1.99 2.09 2.19 2.68 1.90° 2 . 0 8 b - ° 2 . 3 1 b 2 . 8 3 a 1.12 .80 1.09 1.04 .40 a .26b .29b . 3 1 b 4 3 b,c Aih .36° .55" 1.02° 1.17 b ' c 1.25 b 1.46 a ,39b .40b .44b ,50 a ,36b . 3 6 b .43a .33b 4.71 4.74 4.99 8.15 .95 .87 1.01 1.10 .15 .21 .21 .19 .60b .63b . 6 5 b .83" .45b . 5 7 a ' b . S7 a .l' . 7 3 a 1.19 1.31 1.29 1.61

NS 0.050 NS NS 0.050 0.010 NS 0.050 0.010 0.050 NS 0.005 0.005 0.005 0.050 0.050 NS NS NS 0.050 0.050 NS

1 Total free amino acids exclude glutamic acid, methionine sulfoxide, ammonia and ethanolamine. Glutamic acid was excluded from this group because its high concentration masked the change in concentration of the other free amino acids. 2 All free amino acid and amino compound values are in /iM./ml. a b,c - Values not followed in the same horizontal line by the same letter are significantly different.

total amino acids and 121.25 jJiM./ml. total calculated free amino compounds were found. Eleven other compounds were detected but not quantitated because: (a) they were present in trace quantities; (b) or they were not identified; (c) or they were not adequately separated. Glutamic acid accounted for 70.8% of total free amino acids and 66.6% of total amino compounds. Glutamic acid, aspartic acid, threonine, serine, alanine, glycine and arginine comprised 91.1% of total amino acids with the other 10 free amino acids providing only 8.9% of the total. These figures are generally higher than those reported by Graham et al. (1964) and Ahluwalia and Graham (1966) from replicate pooled samples; however, the present study indicated that several factors influence seminal amino acid content. The effects of frequency of collection on

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Volume (ml.) Total sperm X10» Sperm concentration XlOVml. Total free amino acids 1 , 2 Methionine sulfoxide Aspartic acid Threonine Serine Glutamic acid a Amino adipic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ammonia Ethanolamine Ornithine Lysine Histidine Arginine

semen production and free amino acid and amino compound contents are shown in Table 4. Samples collected at 2 day intervals (Interval 4) were significantly higher than the other 3 frequencies in total free amino acids, a-amino adipic acid, tyrosine, phenylalanine, and lysine content (P < 0.05) than Interval 1 and 2 samples. Histidine content of Interval 4 samples was higher than Interval 1 (P < 0.05). Alanine (P < 0.05), isoleucine and leucine (P < 0.005) were significantly higher in Interval 4 samples than the other 3 intervals and higher in Interval 3 than in Interval 1 samples. There were no significant differences in volume, total sperm or sperm cell concentration with different collection frequencies. This agrees with the findings of Brown (1959). The second 7 day interval period of collections was not sufficient time for free amino acid content to return to the initial 7 day interval level. The gradual rise in free amino acid content with the increased frequencies is unlikely to be due to seasonal change or age of the torn because this conflicts with the results of our seasonal change study. The data showing linear, quadratic and cubic regressions of semen production and free amino acid and amino compound content over a 20 week period of the breeding season are presented in Table 5. While volume (P < 0.01), average temperature and ornithine increased (P < 0.005), total free amino acids, methionine sulfoxide, serine, glycine, alanine, valine, methionine, isoleucine, leucine, ethanolamine, ammonia, lysine (P < 0.005) and phenylalanine (P < 0.01) decreased significantly over the 20 week collection period. Correlation analysis of this data showed a positive and highly significant relationship between temperature and volume (r = .677) and a negative relationship between temperature and total free amino acids (r = —.503), serine (r = —.499), methionine (r =

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FERTILITY OF TURKEYS TABLE 5.—F values for regressions of volume, average temperature, spermatozoa concentration and some turkey seminal plasma constituents from 10 birds collected weekly for 20 weeks Linear F

Volume Ave. temp. Sperm cone. Total free amino acids1 Methionine sulfoxide Aspartic acid Threonine Serine Glutamic acid a-Amino adipic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ammonia Ethanolamine Ornithine Lysine Histidine Arginine

9.005** 29 122*** NS 24.712*** 12.896*** NS NS 39.144*** NS NS 37.101*** 36.193*** 12.440*** 27.769*** 30.808*** 13.351*** NS 9.217** 171.184*** 24.824*** 16.048*** 31.647*** NS 6.070*

Quad F 6.271* 17.316*** NS 17.024*** 10.351*** H 99*** NS 28.970*** NS 2.944* 17.463*** 21.435*** 7.783*** 14.856*** 17.802*** 11.807*** NS 6.018* 85.003*** 14.722*** 10.141*** 15.330*** 5.772* 3.040*

Improvement NS NS NS NS 4.870* 20.603*** NS 6.389* NS 5.028* NS NS NS NS NS 6.189* NS NS NS NS NS NS 6.376* NS

Cubic F 6 947*** 15l162*** NS 11.550*** 7.351*** 10.053*** NS 26.642*** NS NS 13.447*** 27.764*** 6.114** 9.330*** 11.446*** 7.572*** NS 3.852* 60.620*** 11.164*** 6.514*** 13.360*** 4.150* NS

Improvement 4.93* NS NS NS NS NS NS 5.541* NS NS NS 11.719*** NS NS NS NS NS NS NS NS NS NS NS NS

+ or slope

+ + — —

-+ — — — — — — — — — — —

+ —

— —

* Significant at P <0.05. ** Significant at P < 0 . 0 1 . *** Significant at P<0.005. 1 Total free amino acids exclude glutamic acid, methionine sulfoxide, ammonia and ethanolamine.

— .519), isoleucine (r = —.472), and histidine (r = —.514) at P < 0.05 and ammonia (r = —.593) at P < 0.01). Volume was negatively correlated with total free amino acids (r = —.535), methionine sulfoxide (r = —.459), serine (r = —.459), glycine (r = —.552), alanine (r = -.539), valine (r = - . 4 7 0 ) , phenylalanine (r = —.513), ethanolamine (r = — .548), ornithine (r = —.571) and lysine (r = -.531) at P < 0.05, isoleucine (r = -.522) at P < 0 . 0 1 , methionine (r = — .628) and ammonia (r = —.729) at the P < 0.005 level of significance. Sperm cell concentration was positively correlated with phenylalanine (r = .534) at the P < 0.05 level of significance. Correlation analysis of the data obtained from the 65 individual torn samples revealed no significant relationships between fertility, percent hatch of eggs set, or sperm

concentration with any free amino acid or amino compound analyzed. The correlation values were quite low and randomly negative and positive. Aspartic acid was approaching significance at the 5% level (r = .244). Fertility was significantly correlated with percent hatch of eggs set (r = .263) at P < 0.05. Analysis of variance of the 65 samples with fertility data and the 22 samples from birds that went out of semen production before use in artificial insemination also revealed no significant difference in free amino acid and amino compound content. Dividing the 65 samples with fertility data into 3 groups according to fertility range and comparing these groups with the no fertility data group also revealed no significant difference. Experiment 2: Chemistry of the Female

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Variable

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TABLE 6.—Means and differences in inorganic materials per 100 grams dry weight from different areas of 28 female turkey hen reproductive tracts

v S

Pmg. K mg. Ca mg. N a mg. Mg mg.

829.9 b * 1042.9 s ** 127.8°** 1042.2"** 87.0 b **

Isthm

-

1072.1"** 528.6b" 257.0 b ** 502.0 b ** 65.0°

««"»» dibtrm 782.6 b ° 581.2 b ** 59. l d 323.0° 112.3 a **

Total major minerals mg. 3129.8

740.8° 0.25° 342.9 s ** 521.8 b ** 85.6 b ** 1682.4

Trace minerals Sr P.p.m. Fe P.p.m. Zn P.p.m. Cu P.p.m. Mo P.p.m. M n P.p.m. B P . p.m.

0.80° 118.2°** 126.1 s ** 23.9°* 0.45d 0.37 b 3.33b"

Total Trace Minerals P.p.m. 273.15

5.26 b * 155.5 b ** 71.6 b ** 38.7 b * 2.39 s ** 5.11 s ** 2.89 b **

0.17° 57. l d 41.2° 6.5d 0.87°*' 0.70b 1.39°

27.40 s ** 223.6 s ** 112.4 s ** 110.9 s **

281.45

107.93

485.95

i.85s** 5.33 s **

* Significant at P <0.05 ** Significant at P <0.01 i*,b,c,d Values not followed in the same horizontal line by the same letter are significantly different.

Reproductive Tract. The mineral content of tissue in the utero-vaginal area, the isthmus, magnum and the infundibulum of female turkey reproductive tracts are found in Table 6. There was no difference in mineral content of high and low fertility hens within individual areas of the reproductive tract, but there were many significant differences in mineral content between areas. The utero-vaginal area contained the highest amount of total major minerals while the infundibulum had the lowest concentration. However, the infundibulum had more total trace minerals than the other areas of the tract. The differences in the calcium, sodium, strontium and potassium content of different areas were very striking. The calcium level in the infundibulum was nearly six times that of the magnum. This high level of calcium in the infundibulum, the site of fertilization, was surprising because seminal plasma calcium was negatively correlated with fertility. Sodium content of the utero-vaginal area was over three times greater than that found in the magnum. There was 34 times as much

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S

strontium in the infundibulum as in the magnum. However, these differences were not nearly as pronounced as the differences in potassium content where the utero-vaginal area contained over 4170 times more potassium than the infundibulum. A repetitive pattern was seen in manganese, molybdenum and strontium contents which were low in the utero-vaginal area and magnum and in high concentrations in the isthmus and infundibulum. The enzyme activities and reducing substance contents of the utero-vaginal area, infundibulum, thigh muscle and blood serum from normal female turkey hens are found in Table 7. There were no significant differences in enzyme activity or reducing substance content between high and low fertility hens. However, there were significant differences between tissues. Glutamic oxalacetic transaminase (GOT) activity of blood serum was over forty times lower than that of thigh muscle and over 160 times lower than that of the infundibulum and utero-vaginal tissue (significant at P < .01). Lactic dehydrogenase (LDH) activity of utero-vaginal and infundibular tissue was significantly higher (P < .01) than LDH activity of thigh muscle or blood serum. The glandular tissue of the former 2 areas may have been actively producing LDH. Alkaline phosphatase activity of blood serum was ten times higher than that of thigh muscle and over three times higher than utero-vaginal and infundibular tissue (significant at P < .01). The alkaline phosphatase activities of the last three tissues may be partly due to the circulatory system elements in these tissues with the glandular tissues having a richer supply of blood vessels. Acid phosphatase activity was significantly higher (P < .01) in infundibular tissue than in the other two tissues or in blood serum. This suggests that the infundibulum may be actively producing acid phosphatase. Gilbert

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FERTILITY OF TURKEYS TABLE 7.—Means and differences in enzyme activity and reducing substance content of 22 female turkey hen tracts, thigh muscle and normal female turkey Wood serum Utero-vaginal 1 3,209.6 b ** 16,431.8'** b

8.79 ** C

180.9 * 7.61 b 220.6"

5,246.8 b ** 15,927.3'** b

8.28 ** 533.2'*** 8.78 b 190.9'

Thigh muscle1 8,590.9"** b

ll,536.4 ** 2.37° b

275.2 * 97.98'** 271.7 b *

Blood serum2 203.7" 99.4° 25.57'** 53.4 d 24.65 b 311.5'*

1

Units per one gram wet weight of tissue. Units per one ml. blood serum. * Significant at P<0.05. ** Significant at P<0.01. a.b.o.d Values not followed in the same horizontal line by the same letter are significantly different. 2

cl al. (1968) reported that sperm host glands in the chicken hen secrete acid phosphatase. Cholinesterase activity of thigh muscle was significantly higher (P < .01) than that of the other two tissues or that of blood serum. Reducing substance content of blood serum was significantly higher (P < .05) than that of the other three tissues and thigh muscle had a significantly higher (P < .05) reducing substance content than the utero-vaginal or infundibular tissues. Gilbert et al. (1968), using histo-chemical techniques, reported no carbohydrate or only mucopolysaccharides were found in the female chicken uterine glands. SUMMARY A positive and significant correlation (P < .01) was found between percent fertility and percent hatch. Negative correlations (I' < .01) were found between both percent fertility of semen and percent hatch with both seminal chloride and sodium. Calcium was negatively correlated (P < .05) with percent fertility while phosphate was positively correlated (P < .05) with percent hatch. In general, seminal amino acid and amino compound content increased with increasing frequency of collec-

tion while the content decreased over a 20 week collection period, with the exception of ornithine which showed an increase. Semen volume and ambient temperature also increased over this period of time. There were no significant correlations between percent fertility or percent hatch of total eggs with any of the free amino acids or amino compounds studied. Also, there were no significant differences in free amino acid and amino compound content between birds that remained in semen production and those that went out of semen production before fertility data was obtained. There were no differences found in the organic or inorganic substances analyzed between samples taken from high fertility hens and low fertility hens. However, there were many differences observed between chemical composition of the different areas of the female reproductive tract from which the samples were taken. The uterovaginal area had the highest content of K, Na and Zn. The isthmus had the highest content of P, Mo and Mn while the magnum had the highest content of Mg and the infundibulum was highest in Ca, Sr, Fe, Cu and B. Many differences were also found between areas and organic constituents. Thigh mus-

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G.O.T. I.U. LDH I.U. Alkaline Phosphatase K.A. units Acid Phosphatase K.A. units Cholinesterase Cholinesterase units Reducing Substances mg%

Infundibulum1

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GRAHAM, SCHMEHL, BROWN, CRABO AND ERTEL

REFERENCES Ahluwal : a, B. S., and E. F. Graham, 1966. Free amino acids in the semen of the fowl and the turkey. J. Reprod. Fert. 12: 36S-368. Al-Hakim, M. K., E. F. Graham and M. L. Schmehl, 1970. Free amino acids and amino compounds in bovine seminal plasma. J. Dairy Sci. S3: 84-88. Babson, A. L., P. O. Shapiro, P. A. R. Williams and G. E. Phillips, 1962. The use of a diazonium salt for the determination of glutamic oxaloacetic transaminase in serum. Clin. Chim. Acta, 7: 199-205. Brown, K. I., 1959. Electrolyte composition and freezing point depression of turkey seminal fluid. Poultry Sci. 38: 804-806. Burriel-Marti, F., and J. Ramirez-Munoz, 1957. Flame Photometry. Elsevier Pub. Co., New York. Dunklegod, K. E., 1968. Relationship of free amino acids in the plasma to dietary requirements of the turkey. Ph.D. Thesis, University of Minnesota. El Jack, M. H., and P. E. Lake, 1967. The content of the principal inorganic ions and carbon dioxide in uterine fluids of the domestic hen. J. Reprod. Fert. 13: 127-132. El Zayat, S., and A. van Tienhoven, 1961. Effect of chloride ions on cock spermatozoa. Amer. J. Physiol. 200: 819-823. Fiske, C. H., and Y. Subbarow, 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66:375-400. Fujii, S., T. Tamura and H. Kunisaki, 1965. Histochemical study of mucopolysaccharides in goblet cells of the chicken oviduct. J. Fac. Fish. Anim. Husb., Hiroshima Univ. 6: 25-35.

Gilbert, A. B., M. E. Reynolds and F. W. Lorenz, 1968. Distribution of spermatozoa in the oviduct and fertility in domestic birds. V. Histochemistry of the uterovaginal sperm-host glands of the domestic hen. J. Reprod. Fert. 16: 433444. Graham, E. F., R. H. Schultz, L. A. Johnson and M. L. Fahning, 1964. A comparison of the free amino acids in seminal plasma of the bull, boar, cock, turkey and uterine fluids of the cow during estrus. V. Intern. Congr. Anim. Reprod. and A. I., Trento, Italy, 4 : 381-386. Hamilton, P. B., 1962. Ion exchange chromatography of amino acids—Microdetermination of free amino acids in serum. Ann. New York Acad. Sci. 102: 55-75. Hoffman, W. S., 1937. A rapid photo-electric method for the determination of glucose in blood and urine. J. Biol. Chem. 120: 51. Kessler, G., and M. Wolfman, 1964. An automatic procedure for the simultaneous determination of calcium and phosphorus. Clin. Chem. 10: 686703. Lake, P. E., 1960. Studies on the dilution and storage of fowl semen. J. Reprod. Fert. 1: 3035. Lake, P. E., 1962. Phosphorus compounds in fowl spermatozoa. Proc. X I I World's Poultry Cong., 105-108. Lake, P. E., 1966. Physiology and biochemistry of poultry semen. Adv. Reprod. Phys. Vol. 1, Logos Press LTD. London. Lake, P. E., E. J. Butler, J. M. McCallum and I. J. Maclntyre, 1958. A chemical analysis of the seminal and blood plasma of the cock. Quart. J. Exptl. Physiol. 43 : 309-313. Lake, P. E., and M. H. El Jack, 1964. Further observations on the chemical composition of the seminal plasma of the domestic cock. Proc. V. Intern. Congr. Anim. Reprod. and A.I., Trento, Italy, 11:359-363. Lake, P. E., and M. H. El Jack, 1966. The origin and composition of fowl semen. Physiology of the Domestic Fowl. Oliver and Boyd Ltd. Edinburgh. Lake, P. E., and M. Hatton, 1968. Free amino acids in the vas deferens, semen, transparent fluid and blood plasma of the domestic rooster, Galium domesticus. J. Reprod. Fert. 15: 139-143. Lake, P. E., and W. M. Maclndoe, 1959. The glutamic acid and creatine content of cock seminal plasma. Biochem. J. 7 1 : 303-306. Levy, A. L., C. Dalmasso and J. Daly, 1965. Technicon Symposium, Automation in Analytical Chemistry, New York: 551-554.

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cle was high in GOT and cholinesterase activities while the utero-vaginal and infundibular tissues were high in LDH activity. Blood serum was high in alkaline phosphatase activity and reducing substance content. Infundibular tissue also had a high acid phosphatase activity. There is still much to be learned concerning female reproductive tract chemistry and its relationship to fertility and maintaining the fertilizing capacity of spermatozoa. At this point, the little knowledge of this relationship is inconclusive.

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Methodology N-14b. Technicon Instruments Corp., 511 Benedict Ave., Tarrytown, N.Y. 10591. Tagnon, H. J., and A. Steens-Lievens, 1960. Inactivation of acid phosphatase in human prostatic homogenates in vitro: Role of calcium. Nature, 185: 48-49. Takeda, A., 1959. Studies on the cock semen. 1. Na, K and Ca levels on the seminal plasma. Res. Rep. Fac. Text. Sericult Shinshu Univ., 9: 55-59. Tamura, T., and S. Fujii, 1966. Histological observations on the secretions of the glands and the mucous cells. J. Fac. Fish. Anim. Husb. Hiroshima Univ., 6: 373-393. Wales, R. G., T. W. Scott and I. G. White, 1961. Biuret reactive materials in semen. Australian J. Exp. Biol. Med. Sci. 39: 455-462. Weichselbaum, T. E., 1946. An accurate and rapid method for the determination of proteins in small amounts in blood serum and plasma. Amer. J. Clin. Path. 7 : 40. Wilcox, F. H., and H. R. Wilson, 1961. The effect of the addition of potassium, magnesium and chloride ions to the diluent used in storing chicken semen. Poultry Sci. 40: 701-704. Winter, G. D., 1960. Cholinesterase activity determination in an automated analysis system. Ann Arbor, New York. Acad. Sci. 87: 629-635. Zall, D. M., D. Fisher and M. O. Garner, 1956. Photometric determination of chloride in water. Anal. Chem. 28: 1665.

Effect of Insulin Injections Upon Plasma Free Fatty Acids (F.F.A.) and Lipolysis of Adipose Tissues of White Leghorn Hens V. PETER* AND S. LEPKOVSKY Department of Poultry Husbandry, University of California, Berkeley, California 94720 (Received for publication January 11, 1971)

I

NSULIN, parenterally administered into mammalian species leads to a fall of plasma free fatty acids (F.F.A.) (Engel, 1962; Vaughan and Korn, 1962). In contrast, administration of insulin into domes* Present address: Poultry Research Institute, Ivanka pri Dunaji, Bratislava, Czechoslovakia.

tic fowl leads to an increase in plasma F.F.A. (Heald et al, 1965; Lepkovsky et al, 1967). Heald et al. (1965) suggested that the action of insulin was due to a glucagon impurity since adipose tissue of the fowl is very responsive to treatment with glucagon (Grande, 1970; Heald et al, 1965). That the increase in plasma F.F.A.

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Lorenz, F. W., 1959. Reproduction in the domestic fowl: Physiology of the male. In: Reproduction in Domestic Animals. Academic Press, Inc., New York. 2 : 343-398. Mann, T., 1964. Biochemistry of Semen and of the Male Reproductive Tract. Methuer and Co., Ltd. London. Marsh, W. A., B. Fingerhut and E. Kirsch, 1959. Adaption of an alkaline phosphatase method for automatic colorimetric analysis. Clin. Chem. 5: 119-126. Powell, M. E. A., and M. J. H. Smith, 1954. The determination of serum acid and alkaline phosphatase activity with 4-amino-antipyrine. J. Clin. Path. 7 : 245-248. Pytasz, M., and B. Klymiuk, 1961. Proteins and non-protein nitrogen in the semen of the domestic fowl. Med. Wet. 17: 437-440. Schindler, H., R. Volcani and S. Weinstein, 1958. Changes in pH during storage, buffering capacity, and glycolysis of cock and bull semen. Poultry Sci. 37: 21-23. Skeggs, L., 1965. Technicon Auto Analyzer Methodology N-5b. Technicon Instruments Corp., 511 Benedict Ave., Tarrytown, N.Y. 10591. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York. Steens-Lievens, A., and H. J. Tagnon, 1962. Reversible inactivation of acid phosphatase in human prostatic fluid. Nature, 195: 400. Stevens, D. L., 1965. Technicon Auto Analyzer

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