Sorting semen may be advantageous The perspectives of sexed bull semen are wide and obvious for cattle breeding:

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1 Sexed semen is a dream coming through Head of Research and Development Søren Borchersen, A.I. Centre Dansire -address: SOB@dansire.dk Bilag til konference i Dnepropetrovsk The use of sexed semen in the cattle breeding works under Danish conditions. The perspectives of sexed semen are wide and obvious in cattle breeding. The company XY Inc. in Texas holds the worldwide patent for sorting semen using flow cytometry. Today this method is the only method used for commercial production of sexed semen. New Danish figures for 9 Danish bulls from the three main dairy breeds in Dansire show in average conception rates below 50 percent for heifers. This is slightly lower than when using conventional semen, however still very interesting when used correctly in praxis. At the same time the results show a high reliability of the sex from semen of the two bulls at more than 90 percent born heifer calves. At the same time the investigation shows that the survival rate of the calves is normal. The results are in fine correspondence with many minor, foreign insemination trials of sexed semen. The Danish investigation is the first major investigation, in which the inseminations were carried out by A.I. technicians in more than 60 ordinary dairy herds. Sorting semen may be advantageous The perspectives of sexed bull semen are wide and obvious for cattle breeding: Increased breeding progress also for the functional traits; udder health, resistance to other diseases, calving ability and fertility in the individual herd Possibility of easier calving and an increased number of live born calves when using heifer semen (semen carrying the x chromosome) for heifers, because the heifers have higher calving ease when giving birth to heifers and the heifer calves have a higher survival rate than born bull calves. The production of beef cattle cross breed calves within dairy cattle (when using beef cattle sires for the rest of the cows and heifers) may increase the production of quality beef. The herd can obtain the desired heifer calves and avoid destruction of undesired bull calves. The disadvantages are the higher price per semen dose; that there is no 100% guarantee of the sex using this method and also the increased number of returns of each pregnancy, which could result in a higher number of days open. However, this will be compensated for abundantly by the financial and production related advantages of sexed semen in the form of better prices of beef cattle and increased genetic progress in the herd. In case of a herd enlargement it is possible to take advantage of sexed semen. The herd can have more heifers from own breeding and in this way avoid purchasing cattle and the following risk of infection. When having a surplus of heifers, it will be possible to discard a number of the bad cows of the herd. An average SDM herd with 100 animals may make 13,000 DKK (1,700 Euro) yearly and a similar Jersey herd may make an extra profit of 63,000 DKK (8,400 Euro) yearly in the form of extra breeding progress and larger beef production when using sexed semen (1).

2 The technological Development Since the end of the 80 s it has been possible to sort semen using flow cytomation so that the following insemination could result in the desired sex (2). Despite many years of research in various methods of sorting semen with X and Y chromosome, at present time flow cytomation is the only method with a high rate of reliability of the sorting (3). Until 1998 the speed of the sorting procedure was approx. 100 sperm per second. Developing the high-speed flow cytomation for sorting made it possible to sort approx. 1,500 sperm per second (4, 5). This lead to a renewed interest in the possibility of commercial utilization of sexed semen, and the speed of the sorting procedure has now increased to approx. 5,000 sperm per second due to the possibility of obtaining better separation of the sperm (3). The company XY Inc., Colorado, USA, was established in XY Inc. has bought the relevant patents within this field and has entered into cooperation with DakoCytomation, which now produce a special type of high-speed flow cytometric sorting of semen. XY Inc., Colorado, is May 2007 acquired by Sexing Technologies, Texas, USA. Since 1998 the company has entered into cooperation projects with A.I. centres and research institutes in among others the USA, Argentina, Germany, Japan, Switzerland, UK, Australia and latest in China. Reports of more than 10,000 born calves from this method have been made. The fertility using sexed semen is still not at the same level as when using conventional semen (3). The lower rate of fertility could be caused by the lower insemination dose used in connection with sexed semen (6). Other reasons for the lower rate of fertility when using sexed semen could be damage to the sperm when sorting or in connection with the colouring (7). In general all trials show that the main reason for lower fertility is the lower sperm concentration in the semen dose two million sperms per straw for sexed semen compared to million in a conventional semen dose. Since 1995 Denmark has invested in the development of new and more precise methods to describe the fertility ability of the semen (8, 9, 10). To a higher degree these methods in connection with sexed semen will early in the process make it possible to identify the semen that does not tolerate the sorting process and the following freezing. At the same time the methods can be used to improve the current protocol for sorting and the following treatment and freezing of the semen. The first Danish results from a major, Danish test of dilution show differences in how the bulls are suited for fertilization (11). The Method of Flow Cytomation The DNA content of the sperm is coloured with a fluorescent stain Hoechst, (12). The sperm alive is stained for one hour so that the stain reflects the DNA content of the sperm. The X sperm stained proportionally fluoresces more than a Y sperm because sperm carrying the X chromosome contain 3-4 percent more DNA than the Y sperm. When the flow cytometry has registered the flourescense, the data is processed in a computer and the liquid is separated into drops using a very exact vibration of the tip of the jet pipe. Then the drop is charged. The X sperm is charged negatively and the Y sperm is charged positively (3). At the tip of the pipe the sperm moves towards the two charged poles depending on its sex and is separated in an X population and a Y population, respectively. In principle the sperm is weighed and separated into three piles a pile of female sperm, a pile of male sperm and a pile of sperm which could not be determined to be male or female. Sorting the sperm has been directed at cattle and horses primarily due to the commercial interest. However, the interest is caused by the wide use of artificial insemination of horses and cattle and

3 the used insemination technique. As regards pigs, an insemination dose is 200 times the size of the dose used for cattle. Therefore sorting a dose of semen for pigs would take long time and make high demands on the insemination technique. New investigations show that the difference of the DNA content between the X and the Y sperm are not the same for the European cattle breeds and the Brahman in Asia (13). The investigation also shows that there is a larger difference in the DNA content for X and Y chromosome sperm in sperm from Jersey bulls than in e.g. bulls of the Holstein Frisian, Hereford and Angus breeds (13). A larger difference in the DNA content makes the sorting easier. The efficiency of sorting is dependant on the individual stages of the sorting process. When sorting the efficiency is typically 22 percent corresponding to the fact that 11 percent X sperm and 11 percent Y sperm are collected while the rest are lost (3). In a normal dose of bull semen approx. 10 percent sperm are dead (14). A part of the sperm is lost due to the staining, sorting and freezing processes. Having sorted the sperm using the flow cytometry, the X and Y sperm are gathered in a Tris egg dilution (15). The sperm that has been sorted is concentrated and diluted in the Tris egg dilution with 2 mill. X sperm per insemination dose. It is important that the semen has been packed precisely in order to ensure the content of the insemination dose (13). Sexed bull semen has been frozen successfully (13, 19). Due to the speed of the sorting process of 5,000 sperm per second, one dose of sexed bull semen contains only approx. 2 mill. sperm compared with mill. sperm in an insemination dose of conventional semen. Results of inseminations with sexed semen in Danish cattle herds Since May it has been possible to use sexed semen in Danish dairy herds from bulls within the three main dairy breeds Red Danish Dairy Breed (RDM), Danish Holstein (SDM DH) and Danish Jersey (DJ). Dansire started producing sexed semen for trials in June The first trial with three bulls was carried out with Danish Holstein from August until January The second trial with three bulls was carried out with Danish Jersey (DJ) from October until April And the third trial with three bulls was done with Red Danish Dairy Breed, RDM, from December until May The trials for all breeds were conducted in herds per breed in total 62 herds. Per herd were used equal doses of sexed semen from the three bulls and equal doses of conventional, control semen. In total minimum 150 doses of sexed semen and minimum 50 doses of control doses per bull were used. The semen was only used on heifers. The results with the three bulls were handled by drawing data from the Danish Cattle Database covering the period August 2006 to September 2007 (16). The average conception rate for conventional bull semen (10-15 mill. Sperm per dose) for all cows and heifers is 38 percent in Denmark. Corresponding to 2.5 dose per pregnancy. The normal pregnancy rate for heifers is expected to be around 60 percent. An Israeli study has made up the conception rate for conventional semen to 64.6 percent for heifers (1.6 doses per pregnancy), for 1 st calf cows 43.0 percent (2.3 doses per pregnancy), for other cows 36.4 percent (2.7 doses per pregnancy) (18). This illustrates the biological differences depending on the age of the females and shows the declining conception rate with an increasing number of lactations. The results from the Danish trial are showed in the following table. IT IS IMPORTANT TO UNDERLINE THAT THE RESULTS ARE PERCENT PREGNANCY NOT NON RETURN

4 RATE the NRR, Non Return Rate, is approximately percent higher. The difference of the statement of the conception rate and non return rate, 0-56 days is illustrated with the different figures. The positive pregnancies in the table have been counted as pregnancies shown by pregnancy test by a technician palpation. The non-pregnancies have been made up from negative pregnancy tests. The non-return rates have been made up for 0-56 days compared with 1 st inseminations and contain therefore no information on the pregnancy result after 56 days. There is a high correlation between the conception rate and the non-return rate. In general the conception rate underestimates the fertility, since it does not include any pregnant animals which are no longer in the herd. The non-return rate overestimates the fertility since it does not include any abortions after 56 days. The A.I. Centre normally uses the non-return rate due to the fact that it is the first information on the bull`s fertility result. The Danish Holstein breed s average Non-Return Rate, 0-16 days, is 69 percent: 63 percent for cows and 73 percent for heifers (11). But in the mentioned trial we have made the main result by percent pregnancy. The selection of the three bulls is based on breeding values and fertility. In September 2007 the respective breeding values, S-Index (Total merit index) for the three bulls is; Var Elvis: 129, Var Hector: 128 and T James: 120. The first heifer calves are born and the results are promising. More than 90 percent of the new born calves are heifer calves. The next table shows the results per bull for the three Danish Holstein bulls.

5 Conclusion The Danish and International Cattle breeders have great interest in the possibility of using sexed semen. The results show that it is possible to obtain useful results with sexed semen in practice under Danish conditions. The situation will be very similar in other countries with an effective Dairy Cattle population. Sorting semen illustrates that biotechnology sets new standards for the work with the practical breeding. The method may have great influence on the breeding work in the individual herd. Bibliography 1. Borchersen S. Sexed semen options regarding breeding and production in your herd. Danish Cattle Federation Congress, Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 1989; 41: Seidel GE Jr, Garner DL. Current status of sexing mammalian spermatozoa. Reprod 2002;124: Johnson LA, Welch GR. Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency. Theriogenology 1999; 52: Seidel GE Jr. Cran DG, Herickhoff LA, Schenk JL, Doyle SP, Green RD. Inseminations heifers with sexed frozen or liquid semen. Theriogenology 1999; 51: Seidel GE Jr. Fertility of bulls on the edge of the dose-response curve for numbers of sperm per inseminate. Proceedings of 17 th Technical Conference, NAAB 1998: Guthrie HD, Johnson LA, Garrett WM, Welch GR, Dobrinsky JR. Flow cytometric sperm sorting: effects of varying laser power on embryo development in swine. Mol Reprod Devel 2002; 37: Christensen P. Danish Semen Analysis: Fertility vs. Quality Tests. Proceedings of the 19 th Technical Conference on Artificial Insemination and Reproduction, NAAB 2002;

6 9. Hansen C, Christensen P, Stryhn H, Hedeboe AM, Rode M, Boe-Hansen G: Validation of the FACSCount AF System for determination of boar sperm concentration. Reprod Domest. Anim. 2002; 61: Boe-Hansen GB, Avery B, Christensen P, Lehn-Jensen H, Greve T. Sperm Chromatin Structure and IVF in Bulls with Low Fertility in Vivo. Proceedings 28 th Annual Conference of the International Embryo Transfer Society. Theriogenology 2003; 59: Labouriau R, Christensen P, Pedersen J, Borchersen S. Analysis of a pilot experiment on semen dilution for artificial insemination in Cattle. Personlig kommunikation, Johnson LA, Flook JP, Look MV. Flow cytometry of X and Y chromosome-bearing sperm for DNA using an improved preparation method and staining with Hoechst Gamete Res. 1987: 17: Garner DL. Flow cytometric sexing of mammalian sperm. Theriogenology 2005, In press. 14. Christensen P. Personlig kommunikation, Schenk JL, Suh TK, Cran DG, Seidel GE, Jr. Cryopreservation of flow-sorted bovine sperm. Theriogenology 1999: 52: Flagstad P. Danish Cattle Federation, Data draw from the Cattle Database Andersen O. Danish Cattle Federation. Statement of reproduction. Personlig Kommunikation, Israeli annual report: Daniel Hojman: The Dairy Industry In Israel. ISRAEL DAIRY BOARD and ISRAEL CATTLE BREEDERS ASSOCIATION, Thomas CA, Garner DL, DeJarnette JM, Marshall CE. Effect of cryopreservation on bovine sperm organelle function and viability as determined by flow cytometry. Biol Reprod 1998; 58: