GB2031456A

GB2031456A - Method for increasing fertility of mammals

Info

Publication number: GB2031456A
Application number: GB7930342A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1978-09-01
Filing date: 1979-08-31
Publication date: 1980-04-23
Also published as: HU178736B; NL7906539A; FR2435948A1; BE878519A; DD151107A5; ES483763A1; ZA794504B; PT70120A; AU5033979A; IT7925426A0; JPS5547868A; SE7907157L; IL58099A0; FI792718A; NO782997L; IT1207234B; DK360479A; DE2935285A1

Abstract

A method for increasing the fertility of mammals is provided, wherein the mammal is inseminated with semen which before, during or after insemination is mixed with at least one agent which stimulates the immune system of the female. The mixing of the semen with the immune system stimulating agent may be performed by premixing the components prior to artificially inseminating the mammal or may be effected by introducing the agent into the vagina and/or uterus of the mammal before or after artificial or natural insemination. A method for treating semen by mixing it with such an agent and a semen diluent containing such an agent is also provided.

Description

SPECIFICATION Method for increasing fertility of mammals The present invention relates to a method for increasing and improving the fertility of mammals, a method for treating semen to render it suitable for increasing the fertility of mammals when used in artificial insemination and a semen diluent or agent.

In modern animal husbandry and stock-breeding artificial insemination (Al) is the most suitable and economic method of insemination. Many advantageous features are obtained by means of Al; inter alia it is possible with semen from one male to inseminate a substantially larger number of females than would have been possible by normal mating. Semen from males with particularly good hereditary character can be deep frozen and kept in store for a very long period of time and be available for insemination of females when good hereditary characters are desirable in the offspring. Al makes it possible for the individual farmer or breeder to "produce" offspring with desirable properties without any danger of inbreeding and without it being necessary to have one or more "unproductive" males available locally for breeding purposes.

However, it is known that Al results in somewhat lower fertility-rate compared with what can be obtained by natural insemination.

It would be of great economical importance for a breeder if the litter size could be increased above the usual level when using Al for animals giving rise to litters, such as pigs; or if the number of fullborn offspring per artificial insemination could be increased for animals which normally produce only one offspring, such as horses and cows.

Within the field of human medicine is has more recently been thought that variation in fertility can possibly have immunological causes (Beer 8 Billingham, The Immunologi of Mammalian Reproduction (Prenticehall: Englewood Cliffs, N.J.) 1976, Scott J.S. and Jones W.R. Immunology of Human Reproduction, Academic Press, London 1976.) If this assumption is correct it is possible that by means of immunological mechanisms a number of natural phenomena having a direct or indirect bearing on the fertility can be explained. Of such phenomena the following can be mentioned: Mating of animals of different breeds often gives rise to increased fertility, which is genetically termed heterosis, however heterosis does not give any physiological explanation of the phenomenon.If heterosis from a physiological point of view can have an immunological explanation, the immunological response should increase when the animals are not interrelated and hence theoretically one should observe a larger heterosis effect, that is an increase in the fertility.

As is known, mating of related animals results in inbreeding depression and inter alia a decreased fertility. If the physiological cause is immunologically conditioned one should expect a lower immunological response if the animals are more closely related and hence an increased inbreed depression.

Good results can be obtained by using a mixture of semen from several males of the same species. The observed percentage of pregnancy in that case is higher that what could be expected if sperms from the most fertile of the males had been used. However, from a breeding point of view it is not advantageous to use mixtures of semen from different males of the same species as one cannot predict which of the males will be biological father of the offspring.

Comparison of litter size after natural mating with litter size after use of Al for pigs clearly shows, as previously mentioned, that smaller litter sizes result from use of Al. If the cause is that the ejaculate contains important antigens the explanation for this might be that the strong dilution of the semen results in smaller litter size. Investigation in connection with hybrid pigs in Norway has shown that with use of artificial insemination very little heterosis for litter size is observed, whereas the corresponding heterosis effect is nearly 1.0 piglet with natural mating.

The physiological explanation might be as indicated above. It has also been shown that the male pig has a significant influence on the litter size. If there exists a correlation between immune response and litter size (embryonic death), it is good reason to presume that the parents will have an influence on the litter size.

The present invention accordingly provides a method for increasing the fertility of a mammal, wherein the mammal is inseminated with semen which before, during or after insemination is mixed with at least one agent which stimulates the immune system of the female, and also provides a method for treating semen which comprises mixing the semen with at least one agent which stimulates the immune system of the female.

Tests have shown that, by employing the method of the present invention one can obtain improved fertility, for example, if the agents which stimulate the female immune system, e.g.

antigens, are mixed with semen, particularly if the semen is diluted with a diluent, prior to artificial insemination with this semen dose. The diluent is preferably used in order to dilute the ejaculate to provide a large number of insemination doses and the diluent can, for example, consist of different buffer mixtures further containing agents to reduce the temperature shock effect if the doses are to be cooled or deep frozen, further antibiotics, nutrients etc. The diluent can also be based on egg yolks, skimmed milk, dry milk etc.. The use and composition of diluents for semen are well known within the state of art and in this respect reference is made to the classic text book of E.S.E. Hafez, Reproduction in Farm Animals, pp. 153-154.Lea 8 Felsiger, Philadelphia 1 966. Further reference is made to Palson Einarsson, Allanbane Insemination och Insemination Teknik Centraltrykkeriet AB Bores 1 972. From these references it will be apparent that when preparing suitable semen diluent one is aiming to obtain the best living condition for the sperms in order to allow the largest possible number of virile sperms to survive and hence to be able to take part in the fertilisation. The semen diluent or agent containing at least one agent which stimulates the female immune system also forms part of the present invention.

US-Patent 3,718,740 discloses that the motility and fertility of spermatozoa in animal semen preparations are increased when ss-glucorinidase is added thereto. This addition significantly improves motility of sperms after storage. However it has not previously been mentioned or indicated that semen or semen diluted with a diluent can be mixed with agents which positively improve fertility. The aspect of the method of the present invention whereby semen or diluted semen is premixed with agents stimulating the female immune system e.g. antigens enables larger litter sizes to be obtained and improves the chances of obtaining a fullborn offspring per insemination. Suitable antigens are cellular antigens such as leucocytes or lymphocytes.One cannot however add whole blood as the erythrocytes will agglomerate around the sperms and inactivate the same.

In the publication by L.V. Swanson and A.G. Hunter: "Egg Yolk antigens and their effect on fertility in rabbits" (Biology of Reproduction 1, (4) 324-329, 1969), is described experiments to determine which proteins in egg yolk were antigenic, to define them immunologically and to determine if antibodies against egg yolk could effect fertility when mixed with rabbit semen suspended in egg yolk. However it has not previously been suggested that the presence of antigens in semen formulations can increase the fertility.

Another aspect of the method of the present invention comprises introducing the agents which stimulate the female immune system, e.g. antigens, into the mammal's vagina and/or uterus before or after natural or artificial insemination so that the mixing occurs in situ.

By "antigens" in the present invention is understood agents which can stimulate the female immune system and the antigens can be present in form of cellular antigens or "pure antigens" isolated from cells or cell tissue. It is of course necessary to provide the agents or antigens in a form (or number) other than that naturally present in the semen i.e. there must be a positive addition of such agents either to the semen prior to Al or by introducing the agents into the mammal's vagina and/or uterus before or after insemination. In preference, the antigens are used in form of leucocytes or lymphocytes from the same species or from other species, or in the form of foreign cells such as various microorganisms or cells from tissue cultures.It is also possible that a synergistic effect can be obtained by utilising more than one type of antigen from the same species or from different animal species.

An antigen can be defined as any substance stimulating the production of antibodies. Most antigens are proteins, but it is also found that certain other large molecules can serve as antigens. See for instance Stanier, Doudoroff and Adelberg: "The Microbial World" (Prentice Hall, Inc. 1958), pp. 594-871.

The present invention will now be illustrated by the following Examples in which the antigens utilized were leucocytes.

EXAMPLE 1 1. 73 gilts of Norweigian Lard race at weaning age weighing approximately 20 kg. were bought (Spring of 1978).

2. At 2. period of heat the gilts were divided into three groups: A. 1/3 of the gilts were artificially inseminated in the usual manner (control group).

B. 1/3 of the gilts were inseminated with semen to which was added cellular antigens (leucocytes) from the same boar that had produced the semen used for insemination.

C. 1/3 of the gilts were inseminated with semen to which was added cellular antigens (leucocytes) from a different boar of a different breed of pigs (Yorkshire).

3. 4-5 weeks after artificial insemination the gilts were slaughtered and the following were noted: number of corpora lutea number of normal (living) fetuses number of dead fetuses -the size of the fetuses 4. The fetuses were fixed for later determination of the sex ratio and the causes for embryonic death.

The results of this can be summarised as follows: The addition of cellular antigens to the semen resulted in significantly larger litters (a higher number of fetuses) compared with use of the usual sperm doses.

addition of antigens to the semen resulted in a 12% increase in litter size.

-The probability that the observed differences were due to random variation was less than 2.5%.

The averages for the test were: Group Number Normal fetuses in percent Relative of of the number of corpora numbers Animals lutea A control 26 72.57% 100 B 25 83.24% 114.7 C 21 81.88% 112.9 The percentage of normal fetuses in the control group were as normally experienced, whereas the percentages for the groups B and C are much higher than usual.

EXAMPLE 2 In order to verify the promising results of Example 1, 68 gilts (Spring 1979) were divided into 3 groups and arificially inseminated as follows: A. control group, inseminated in the usual way.

B. inseminated with semen mixed with antigens in the form of pooled leucocytes from pigs of Yorkshire breed.

C. inseminated with semen mixed with antigens in the form of pooled leucocytes from Norwegian Red and White cattle.

the fertility rate was determined as previous described Group Number Normal fetuses in percent Relative of of the number of corpora numbers animals lutea A control 24 68 100 B 22 79 116 C 22 77 113 The results from this test substantiate the results obtained in Example 1.

Variance analysis of the results from Examples 1 and 2 gave the following results: 1978 + 1979 average numbers Number of corpus luteum normal fetuses Rel.

Group gilts X a XA a Nos.

Control (I) 50 13.92 2.42 9.30 3.42 100 Leucocytes from the semen donor (11) 25 13.52 2.50 10.69 3.49 114.9 Leucocytes from different breed (III) 44 14.32 2.23 10.53 3.53 113.2 Leucocytes from cattle (IV) 22 14.23 9.93 10.27 3.08 110.4 AThe result is corrected for variation in the number of corpus luteum.

The probability for existence of a real difference in the number of normal fetuses in the control group and the test groups gives the following F-ratios: Not corrected for variation in number of corpora lutea: 4.73** Corrected for variation in number of corpora lute: 5.05** These results indicate that there exists a highly significant (99%) difference between the results obtained for the control group and the test groups.

It is known that the fertility of mammals to a large extent is determined by the rate of fetus death. Premature fetus death is the primary cause for having to rebreed animals. For instance, for horses it has been shown that 100 matings will result in 40 offspring and 24 premature fetus deaths. Ref. E.P. Cunningham, Annual Meeting of the European Association of Animal Production (E.A.A.P.) Stockholm 1 978.

Addition of antigens in accordance with the present invention, to the semen of pigs and cattle has resulted in a significant lowering of premature fetus death and hence increased the fertility.

This can also be expected in the case of horses.

A comprehensive account of the isolation of leucocytes can be found in: Bayum, A. Isolation of Lymphocytes, Granulocytes and Macrophages. Scand. J. Immunol. Vol. 5, Suppl. 5. 1 976.

To a semen dose for pigs, 1 ml of leucocytes obtained in that manner can be added. To a semen dose for cattle can be added approximately 0.5 ml of the antigens and the insemination can be carried out in known manner. As indicated by the experimental results an increase of approximately 12% in the litter size can be obtained, which can be of very great importance.

According to FAO, the world's pig population is approximately 670 mill. of which approximately 60 mill. are breeding sows. If it is assumed that 10% of the sows will be inseminated and further that one can obtain an increase in fertility corresponding to 1/2 piglets per sow, this increase will represent 3 mill. additional piglets per annum. Assuming the value of one piglet to be 50 dollars the total profit on a world basis will be approximately 1 50 mill. dollars per annum.

In countries in which artificial insemination is common an increase in litter size of the order of 12% will be of very great importance.

Many experiments have been performed in order determine the minimum number of sperms required to be inseminated in order to obtain conception. From the diagram in the accompanying drawings reproduced from: G.W. Salisbury, N.L. van Denmark and J.R. Lodge, Physiology of Reproduction and Artificial Insemination of Cattle. W.H. Friedmand 8 Co. San Francisco 1978, it can be seen that the probability of conception decreases rapidly with decreasing number of inseminated sperms. In general, it is assumed that 8 to 1 2 mill. sperms should be inseminated in order to obtain a satisfactory degree of conception, that is, in the range 65 to 68%.

For semen doses of approximately 1 mill. sperms. one would expect at most 4 calves per 10 inseminations. As apparent from the diagram, the X-axis, the Y-axis and the curve are extrapolated in the range below 2 mill. sperms per dose.

EXAMPLE 3 In order to investigate whether addition of antigens could compensate for a reduction in the number of sperms per dose, 10 cows of Norwegian Red and White breed were inseminated with a semen dose containing approximately 1 mill. sperms to which were added 3 X 109 sperms from pig, that is sperm from a different species, which resulted in 9 fullborn calves which is a very good and unexpected result. The bull semen used in this Example was mixed with antigens in the form of boar semen, and pregnancy and fullborn offspring was approximately 90% which greatly exceeds the scale of the above-mentioned diagram.

The fact that the fertility rate can be increased by operating according to the invention is of great economic imporatance. Each percent improvement of this non-return rate in cattle is, according to UB Lindstrm, Nordiske Jordbruksforskeres Kongress i Oslo, July 1979, calculated to be approximately US $0.60 per cow per year.

According to FAO the population of milking cows in the United States, Europe and Russia is approximately 430 mill. In many countries more than 75% of the cows are inseminated artificially, however, assuming that the average for the mentioned countries is 50%, it will mean that approximately 215 mill. cows are inseminated artificially each year.

A large number of artificial inseminations is also carried out in South America, Australia and Asia. In addition, a large number of beef cattle is artificially inseminated each year. However, based on 200 mill. insemination per year an improvement in the conception rate of 2.5% should give a profit of the order of 200 mill. dollars per year.

Further experiments have substantiated that addition of antigens to the semen or to semen diluted with a diluent will increase the percentage of fullborn offspring per insemination. The antigens can be added to the semen per se or to semen diluted with a diluent; however, from a practical point of view it is preferable that the antigen is contained in the diluent or is contained in a composition from which the diluent is prepared by the addition of water. The antigens can also be introduced into vagina and/or uterus before of after natural mating or artificial insemination.

Claims

1. A method for increasing the fertility of a mammal, wherein the mammal is inseminated with semen which before, during or after insemination is mixed with at least one agent which stimulates the immune system of the female.

2. A method according to claim 1 in which the mammal is artificially inseminated with a semen dose to which has been added the agent which stimulates the immune system of the female.

3. A method according to claim 2 in which the semen dose contains semen diluted with a diluent.

4. A method according to claim 1 which comprises introducing into the mammal's vagina and/or uterus, before or after artificial or natural insemination, at least one agent which stimulates the female immune system.

5. A method according to any one of the preceding claims in which the agent stimulating the female immune system comprises antigens.

6. A method according to any one of the preceding claims in which the agent stimulating the female immune system comprises cellular antigens, microorganisms or cells from tissue cultures.

7. A method according to claim 6 in which the cellular antigens are leucocytes or lymphocytes.

8. A method according to any one of the preceding claims in which the agent stimulating the female immune system comprises semen from an animal of another species.

9. A method according to claim 1 substantially as described in any one of the Examples.

10. A method of treating semen which comprises mixing the semen with at least one agent which stimulates the immune system of the female.

11. A method according to claim 10, which the semen is mixed with antigens.

1 2. A method according to claim 10 or 11 in which the semen is mixed with cellular antigens, microorganisms or cells from tissue cultures.

1 3. A method according to claim 1 2 in which the cellular antigens are leucocytes or lymphocytes.

14. A method according to any one of claims 10 to 1 3 in which the semen is mixed with semen from an animal of another species.

1 5. A method according to any one of claims 10 to 14 in which the semen is diluted wtih a diluent.

16. A method according to claim 10 substantially as described in any one of the Examples.

1 7. Semen when treated by a method as claimed in any one of claims 10 to 1 6.

1 8. A semen diluent or agent containing at least one agent which stimulates the female immune system.

1 9. A semen diluent or agent according to claim 1 8 which contains antigens.

20. A semen diluent or agent according to claim 1 8 or 1 9 which contains cellular antigens, microorganisms or cells from tissue cultures.

21. A semen diluent or agent according to claim 20 in which the cellular antigens are leucocytes or lymphocytes.

22. A mammal whose fertility has been increased by a method as claimed in any one of claims 1 to 9 or by insemination with semen as claimed in claim 1 7.