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The Fertility of Fowl Spermatozoa in Glycerol Diluents after Intrauterine Insemination*
RESEARCH NOTES
REFERENCES Carver, D. S., E. E. Rice, R. E. Gray and P. E. Mone, 1954. The utilization of fats of different
melting points added to broiler feeds. Poultry Sci. 33: 1048. Donaldson, W. E., G. F. Combs and G. L. Romoser, 1954. Results obtained with added fat in chick rations. Poultry Sci. 33: 1053. Lewis, R. W., and P. E. Sanford, 1953. Use of equivalent levels of antibiotic and vitamin B ]S supplements with cottonseed and soybean oil meals in chick diets. Poultry Sci. 32: 268-275. Runnels, T. D., 1955. Animal fat in combination with various other ingredients in broiler rations. Poultry Sci. 34:140-144. Sanford, P. E., 1954. Use of waste fat for poultry. (Unpublished research). Kansas Agri. Exp. Sta. Siedler, A. D., and B. S. Schweigert, 1953. Effect of feeding graded levels of fat with and without cholineand antibiotic +B12 supplements tochicks. Poultry Sci. 32: 449-454. Sunde, M. L., 1954. The effects of fats and fatty acids on feed conversion in chicks. Poultry Sci. 33: 1084. Yacowitz, H. 1953. Supplementation of corn-soybean oil meal rations with penicillin and various fats. Poultry Sci. 32:930.
THE FERTILITY OF FOWL SPERMATOZOA IN GLYCEROL DILUENTS AFTER INTRAUTERINE INSEMINATION* T. E. ALLEN AND LIDIA W. BOBR Poultry Research Centre, Werribee, Victoria, Australia (Received for publication June 7, 1955)
It has been well established by Smith and Polge (1950) and Polge (1951), and our experience has been similar, that fowl sperm in diluents giving a final concentration of 15% glycerol are completely infertile whether the semen has been frozen or not unless the sperm suspension is dialysed to effect slow removal of the glycerol prior to insemination. The recommended time of dialysis has been given by Polge (1951) as 2 hours at 20°C. and the technique of insemination he used was * Contribution from Division of Animal Health and Production of Commonwealth Scientific and Industrial Research Organization (C.S.I.R.O.).
that described by Burrows and Quinn (1939). The dialysis is time consuming and damaging to the sperm (if the semen has been deep frozen), therefore a more efficient insemination technique which would increase the fertility of poorly viable sperm samples would be useful since it might render the removal of the glycerol unnecessary. We have modified Burrows and Quinn's method of A.I. to permit the semen to be deposited in the uterus rather than the vagina which is the normal practice. The vagina is a coiled structure, kept in such a position by surrounding fatty tissue,
Downloaded from http://ps.oxfordjournals.org/ at Ritsumeikan University on June 19, 2015
plemented diet was consumed than the basal diet. When the birds in Lot 4 were given a choice between lard and corn oil supplemented diets, the consumption of each diet was approximately equal. Approximately equal amounts of feed consumed by birds in Lot 4, fed both lard and corn oil supplemented diets, resulted in birds with the greatest mean weight of all lots. Birds in Lot 1 fed the K.S.C. basal weighed the least, an average of 1052 grams. Results of this experiment, using palatability preference as evaluated by feed consumption, indicate that chicks prefer diets supplemented with 2% fat.
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RESEARCH NOTES
TABLE 1.—Fertility of pooled ejaculates diluted with a Tyrode-glycerol solution to give a final concentration of 15% glycerol
Normal insemination into vagina Semen deposited in or close to uterus
No. of eggs set
fertile
126
0
116
38
TABLE 2.—Fertility of pooled ejaculates diluted with a Tyrode-glycerol solution to give a final concentration of 15% glycerol
%
fertile
64 66
0 73
%
shows fertility of eggs collected up to one week. Inseminations, using this technique are now being carried out with frozen semen and sperm that are poorly viable for other reasons e.g. in vitro storage. Polge (1951) reported finding no spermatozoa at the top of the oviduct of hens inseminated with semen containing 15% glycerol and that a quick reduction of the glycerol concentration in vitro killed a high proportion of the sperm. He suggested that the infertility might be due to destruction of the sperm by rapid removal of glycerol from the semen in the hen's oviduct. Our experiments confirm his finding that glycerol affects fertility but, as it is unlikely that the glycerol would be removed from the sperm more slowly in the uterus than in the vagina, the infertility is probably due to greatly lowered numbers of functionally intact sperm in the vagina rather than their complete destruction. This in turn would result in lowered numbers of sperm at the upper regions of the oviduct. Using an absolute technique in which a quantitative estimate of sperm numbers can be made by assaying acid digested sections of the oviduct for radioactivity after insemination with P 32 labelled sperm, a technique which will be described in a later paper, much greater numbers of sperm were found in the higher levels of the oviduct after intrauterine insemination than with intravaginal insemina tion. Faeces expelled by
Downloaded from http://ps.oxfordjournals.org/ at Ritsumeikan University on June 19, 2015
but if dissected out its length will be found to be about 8-10 cm. A strong sphincter muscle is present at the junction of vagina and uterus. The insemination syringe we used was similar to that described by Thumin (1951) but a glass cannula about 10.5 cm. long was fitted. Semen was diluted half an hour to one hour previously with an equal volume of Tyrode solution containing 30% glycerol. A volume of 0.2 cc. diluted semen was inseminated. When insemination was performed by guiding the cannula through the coils of the vagina until it was estimated to have entered the uterus, about 50% of inseminated hens laid fertile eggs after insemination. The fertility of eggs collected up to the 7th day after insemination is given in Table 1. Fertility was determined by breaking eggs after 24-30 hours incubation. Higher fertility was obtained more consistently if the insemination was carried out by inserting a forefinger in the vagina and probing for the junction of vagina and uterus and then inserting the glass cannula using the forefinger as a guide. By this method there is no doubt that the semen is deposited in the uterus and if a soft shelled egg is present there the semen can be lodged above it. The method is simple but should be carried out as quickly as possible to avoid a temporary fall in egg production. Almost all birds so inseminated with semen containing 15% glycerol have laid fertile eggs. Table 2
Normal insemination info vagina Intrauterine insemination
No. of eggs set
RESEARCH NOTES
the cervix limits sperm numbers probably differs from that of the utero-vaginal junction of the hen. REFERENCES Braden, A. W. H., 1953. Distribution of sperms in the genital tract of the female rabbit after coitus. Australian J. Biol. Sci. 6: 693-705. Burrows, W. H., and J. P. Quinn, 1939. Artificial insemination of chickens and turkeys. U. S. Dept. Agric. Circ. No. 525. Polge, C , 1951. Preservation of fowl spermatozoa at low temperatures. Proc. IXth. World's Poultry Cong. (3): 11-14. Smith, A. U., and C. Polge, 1950. Survival of spermatozoa at low temperatures. Nature, 166: 668. Thumin, A., 1951. Improvement in the technique of artificial insemination of chickens. Proc. IXth. World's Poultry Cong. (3): 154-158.
PREVENTION OF PREMATURE OVIPOSITION AND SHELL-LESS EGGS WITH EPHEDRINE* DONALD POLIN AND PAUL D. STURKIE Laboratory of Avian Physiology, Poultry Department, Rutgers University, New Brunswick, New Jersey (Received for publication Tune 20, 1955)
Neuromimetic drugs affect retention of the egg within the uterus of the hen, (Weiss and Sturkie, 1952). Ephedrine, the sympathomimetic drug employed by these investigators, causes a delay in oviposition for as much as 36 hours in some cases. This report describes a case in which the drug was used to prevent premature oviposition in one hen that consistently laid eggs without shells. The blood calcium of this hen was found to be 28 mg./lOO ml., which is normal (Polin, 1955). Time of oviposition for four of the eggs was found to be between the hours of 4 to 8 p.m. or soon after the egg reaches the uterus. * Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers University— The State University of New Jersey, Department of Poultry Husbandry, New Brunswick.
Ephedrine sulfate was administered to the hen subcutaneously, in a sesame oil and water emulsion (70/30, vol./vol.) on two separate occasions, about one week apart. At first, 75 mg. of ephedrine sulfate were administered when a soft egg was palpated in the oviduct. The egg was retarded for about 10 hours. Some shell was found on this egg. Two eggs without shells were laid during the interval between treatments. On the second occasion, 100 mg. of ephedrine sulfate were administered, as before. The egg was retarded for approximately 20 hours. The shell and its membrane, after washing and drying, weighed 2.936 grams; the weight of the shell membrane from the previous egg without shell, similarly washed and dried, was 0.827 gram. The shell weight of the treated egg was below normal, even though the length of uterine stay was ap-
Downloaded from http://ps.oxfordjournals.org/ at Ritsumeikan University on June 19, 2015
the birds shortly after intravaginal insemination were found to contain well over 90% of the sperm recovered using the P 32 labelled sperm technique. This technique accounts for about 50% of the total number of sperm inseminated. From these and other experiments which will be published elsewhere it appears that the mechanism of sperm transport in the vagina of the hen differs from that in the reproductive tract distal to the vagina. The utero-vaginal junction, like the cervix in some mammals (Braden, 1953) constitutes a barrier to sperm passage and limits the numbers of sperm reaching the upper parts of the oviduct. Nevertheless the mechanism by which
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REFERENCES Carver, D. S., E. E. Rice, R. E. Gray and P. E. Mone, 1954. The utilization of fats of different
melting points added to broiler feeds. Poultry Sci. 33: 1048. Donaldson, W. E., G. F. Combs and G. L. Romoser, 1954. Results obtained with added fat in chick rations. Poultry Sci. 33: 1053. Lewis, R. W., and P. E. Sanford, 1953. Use of equivalent levels of antibiotic and vitamin B ]S supplements with cottonseed and soybean oil meals in chick diets. Poultry Sci. 32: 268-275. Runnels, T. D., 1955. Animal fat in combination with various other ingredients in broiler rations. Poultry Sci. 34:140-144. Sanford, P. E., 1954. Use of waste fat for poultry. (Unpublished research). Kansas Agri. Exp. Sta. Siedler, A. D., and B. S. Schweigert, 1953. Effect of feeding graded levels of fat with and without cholineand antibiotic +B12 supplements tochicks. Poultry Sci. 32: 449-454. Sunde, M. L., 1954. The effects of fats and fatty acids on feed conversion in chicks. Poultry Sci. 33: 1084. Yacowitz, H. 1953. Supplementation of corn-soybean oil meal rations with penicillin and various fats. Poultry Sci. 32:930.
THE FERTILITY OF FOWL SPERMATOZOA IN GLYCEROL DILUENTS AFTER INTRAUTERINE INSEMINATION* T. E. ALLEN AND LIDIA W. BOBR Poultry Research Centre, Werribee, Victoria, Australia (Received for publication June 7, 1955)
It has been well established by Smith and Polge (1950) and Polge (1951), and our experience has been similar, that fowl sperm in diluents giving a final concentration of 15% glycerol are completely infertile whether the semen has been frozen or not unless the sperm suspension is dialysed to effect slow removal of the glycerol prior to insemination. The recommended time of dialysis has been given by Polge (1951) as 2 hours at 20°C. and the technique of insemination he used was * Contribution from Division of Animal Health and Production of Commonwealth Scientific and Industrial Research Organization (C.S.I.R.O.).
that described by Burrows and Quinn (1939). The dialysis is time consuming and damaging to the sperm (if the semen has been deep frozen), therefore a more efficient insemination technique which would increase the fertility of poorly viable sperm samples would be useful since it might render the removal of the glycerol unnecessary. We have modified Burrows and Quinn's method of A.I. to permit the semen to be deposited in the uterus rather than the vagina which is the normal practice. The vagina is a coiled structure, kept in such a position by surrounding fatty tissue,
Downloaded from http://ps.oxfordjournals.org/ at Ritsumeikan University on June 19, 2015
plemented diet was consumed than the basal diet. When the birds in Lot 4 were given a choice between lard and corn oil supplemented diets, the consumption of each diet was approximately equal. Approximately equal amounts of feed consumed by birds in Lot 4, fed both lard and corn oil supplemented diets, resulted in birds with the greatest mean weight of all lots. Birds in Lot 1 fed the K.S.C. basal weighed the least, an average of 1052 grams. Results of this experiment, using palatability preference as evaluated by feed consumption, indicate that chicks prefer diets supplemented with 2% fat.
1167
1168
RESEARCH NOTES
TABLE 1.—Fertility of pooled ejaculates diluted with a Tyrode-glycerol solution to give a final concentration of 15% glycerol
Normal insemination into vagina Semen deposited in or close to uterus
No. of eggs set
fertile
126
0
116
38
TABLE 2.—Fertility of pooled ejaculates diluted with a Tyrode-glycerol solution to give a final concentration of 15% glycerol
%
fertile
64 66
0 73
%
shows fertility of eggs collected up to one week. Inseminations, using this technique are now being carried out with frozen semen and sperm that are poorly viable for other reasons e.g. in vitro storage. Polge (1951) reported finding no spermatozoa at the top of the oviduct of hens inseminated with semen containing 15% glycerol and that a quick reduction of the glycerol concentration in vitro killed a high proportion of the sperm. He suggested that the infertility might be due to destruction of the sperm by rapid removal of glycerol from the semen in the hen's oviduct. Our experiments confirm his finding that glycerol affects fertility but, as it is unlikely that the glycerol would be removed from the sperm more slowly in the uterus than in the vagina, the infertility is probably due to greatly lowered numbers of functionally intact sperm in the vagina rather than their complete destruction. This in turn would result in lowered numbers of sperm at the upper regions of the oviduct. Using an absolute technique in which a quantitative estimate of sperm numbers can be made by assaying acid digested sections of the oviduct for radioactivity after insemination with P 32 labelled sperm, a technique which will be described in a later paper, much greater numbers of sperm were found in the higher levels of the oviduct after intrauterine insemination than with intravaginal insemina tion. Faeces expelled by
Downloaded from http://ps.oxfordjournals.org/ at Ritsumeikan University on June 19, 2015
but if dissected out its length will be found to be about 8-10 cm. A strong sphincter muscle is present at the junction of vagina and uterus. The insemination syringe we used was similar to that described by Thumin (1951) but a glass cannula about 10.5 cm. long was fitted. Semen was diluted half an hour to one hour previously with an equal volume of Tyrode solution containing 30% glycerol. A volume of 0.2 cc. diluted semen was inseminated. When insemination was performed by guiding the cannula through the coils of the vagina until it was estimated to have entered the uterus, about 50% of inseminated hens laid fertile eggs after insemination. The fertility of eggs collected up to the 7th day after insemination is given in Table 1. Fertility was determined by breaking eggs after 24-30 hours incubation. Higher fertility was obtained more consistently if the insemination was carried out by inserting a forefinger in the vagina and probing for the junction of vagina and uterus and then inserting the glass cannula using the forefinger as a guide. By this method there is no doubt that the semen is deposited in the uterus and if a soft shelled egg is present there the semen can be lodged above it. The method is simple but should be carried out as quickly as possible to avoid a temporary fall in egg production. Almost all birds so inseminated with semen containing 15% glycerol have laid fertile eggs. Table 2
Normal insemination info vagina Intrauterine insemination
No. of eggs set
RESEARCH NOTES
the cervix limits sperm numbers probably differs from that of the utero-vaginal junction of the hen. REFERENCES Braden, A. W. H., 1953. Distribution of sperms in the genital tract of the female rabbit after coitus. Australian J. Biol. Sci. 6: 693-705. Burrows, W. H., and J. P. Quinn, 1939. Artificial insemination of chickens and turkeys. U. S. Dept. Agric. Circ. No. 525. Polge, C , 1951. Preservation of fowl spermatozoa at low temperatures. Proc. IXth. World's Poultry Cong. (3): 11-14. Smith, A. U., and C. Polge, 1950. Survival of spermatozoa at low temperatures. Nature, 166: 668. Thumin, A., 1951. Improvement in the technique of artificial insemination of chickens. Proc. IXth. World's Poultry Cong. (3): 154-158.
PREVENTION OF PREMATURE OVIPOSITION AND SHELL-LESS EGGS WITH EPHEDRINE* DONALD POLIN AND PAUL D. STURKIE Laboratory of Avian Physiology, Poultry Department, Rutgers University, New Brunswick, New Jersey (Received for publication Tune 20, 1955)
Neuromimetic drugs affect retention of the egg within the uterus of the hen, (Weiss and Sturkie, 1952). Ephedrine, the sympathomimetic drug employed by these investigators, causes a delay in oviposition for as much as 36 hours in some cases. This report describes a case in which the drug was used to prevent premature oviposition in one hen that consistently laid eggs without shells. The blood calcium of this hen was found to be 28 mg./lOO ml., which is normal (Polin, 1955). Time of oviposition for four of the eggs was found to be between the hours of 4 to 8 p.m. or soon after the egg reaches the uterus. * Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers University— The State University of New Jersey, Department of Poultry Husbandry, New Brunswick.
Ephedrine sulfate was administered to the hen subcutaneously, in a sesame oil and water emulsion (70/30, vol./vol.) on two separate occasions, about one week apart. At first, 75 mg. of ephedrine sulfate were administered when a soft egg was palpated in the oviduct. The egg was retarded for about 10 hours. Some shell was found on this egg. Two eggs without shells were laid during the interval between treatments. On the second occasion, 100 mg. of ephedrine sulfate were administered, as before. The egg was retarded for approximately 20 hours. The shell and its membrane, after washing and drying, weighed 2.936 grams; the weight of the shell membrane from the previous egg without shell, similarly washed and dried, was 0.827 gram. The shell weight of the treated egg was below normal, even though the length of uterine stay was ap-
Downloaded from http://ps.oxfordjournals.org/ at Ritsumeikan University on June 19, 2015
the birds shortly after intravaginal insemination were found to contain well over 90% of the sperm recovered using the P 32 labelled sperm technique. This technique accounts for about 50% of the total number of sperm inseminated. From these and other experiments which will be published elsewhere it appears that the mechanism of sperm transport in the vagina of the hen differs from that in the reproductive tract distal to the vagina. The utero-vaginal junction, like the cervix in some mammals (Braden, 1953) constitutes a barrier to sperm passage and limits the numbers of sperm reaching the upper parts of the oviduct. Nevertheless the mechanism by which
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