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«Institute of Animal Breeding, Mariensee, FAL, Neustadt, Germany Clinic for Reproduction and Horses, Veterinary Faculty, University of Ljubljana, ...»

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Arch. Tierz., Dummerstorf 49 (2006) 1, 41-54

Institute of Animal Breeding, Mariensee, FAL, Neustadt, Germany

Clinic for Reproduction and Horses, Veterinary Faculty, University of Ljubljana, Ljubljana,



Application of flowcytometrically sexed spermatozoa in different

farm animal species: a review


The most efficient way to shift the sex ratio of offspring is to select spermatozoa according to the sex

chromosomes. The difference of their DNA-content can be used by flowcytometry to produce populations of either sex at high accuracy. As the method is based on single cell identification, the output of cells is limited, although major improvements were made by high-speed flowcytometry and improved cell orientation in front of the UV-Laser. The state of art is described in this review for the main farm animal species. For bovine AI sexsorted-frozen spermatozoa are already available on commercial basis, whereas in other species, especially in pigs, the high demand of spermatozoa can hardly be satisfied. However, in combination with other biotechniques like IVF, ICSI, GIFT and special insemination protocols litters of acceptable size were produced under laboratory condition. As in horses and sheep current research is focused in pigs on long time storage of the sex sorted spermatozoa in liquid nitrogen.

Key Words: sperm sexing, cattle, pigs, sheep, horse Zusammenfassung Titel der Arbeit: Stand und Perspektiven des Einsatzes von durchflusszytometrisch „gesexten“ Spermien bei verschiedenen Nutztierarten – Eine Übersicht Die effizienteste Art, das Geschlechterverhältnis von Nachkommen zu beeinflussen, besteht darin, Spermatozoen entsprechend den Geschlechtschromosomen einzusetzen. Der Unterschied in der DNA-Menge zwischen X- bzw.

Y-chromosomentragenden Spermatozoen kann genutzt werden, um Tierpopulationen jedes Geschlechts mit hoher Sicherheit zu erzeugen. Da die Methode auf der Identifikation einzelner Zellen basiert, ist die Sortierkapazität trotz erheblicher Fortschritte begrenzt. Der Stand der Technik wird für verschiedene Nutztierarten beschrieben. Für die Besamung von Rindern stehen separierte Samenzellen kommerziell zur Verfügung. Demgegenüber können die Anforderungen auf dem Gebiet der Besamung von Sauen mit separierten Spermatozoen derzeit kaum erfüllt werden. Jedoch wurden Würfe akzeptabler Größe in Verbindung mit anderen Biotechniken und speziellen Besamungsprotokollen unter Laborbedingung produziert. Die Forschung wird darauf konzentriert, sortierte Samenzellen der Spezies Pferd, Schaf und Schwein erfolgreich im flüssigen Stickstoff lagern zu können.

Schlüsselwörter: Spermientrennung, Rind, Schwein, Schaf, Pferd Introduction Modern livestock management requires efficient methods to optimize herd size avoiding non or suboptimal producing animals (SEIDEL JR., 2003). Especially, when products are sex related, the opposite sex diminishes farm efficiency. Besides, environmental reasons need to be taken in account showing that high productivity of animals is more beneficial compared to low or average performance, because basic metabolism is almost independent from the level of performance. Therefore, many attempts have been reported over the last decades to modify the sex ratio of offspring.

In the bovine for example, females are required for milk production; whereas males are more preferable for meet production. With sorted spermatozoa, test inseminations KLINC; RATH: Application of flowcytometrically sexed spermatozoa in different farm animal species can be reduced, and calving problems be avoided. In pig production, female end products are preferably requested and hybrid lines would benefit from sex preselection, whereas in companion animals owners demands may differ.

Sex predetermination - historical development Since ancient times, sex determination has been of interest for man. First attempts to control sex have been described by philosophers in pre-ancient Greece.

DEMOCRITUS OF ABADERA (460-370 BC) believed that males originate from the right testicle and females from the left. Further, it was said that males developed more often in the right and females in the left uterine horn. According to this presumption, recommendations for human coitus and in some instances even castration of "unwanted" testis were performed in order to obtain a child of the desired sex. First description of "Sperma" came from HIPPOCRATES (460-377 BC). He believed that "Sperma" plays a key role in the development of a child. If "Sperma", interestingly produced from both sexes, was strong from both parents, then a male would be born.

In opposite "weak Sperma" was correlated with the development of a girl. Different selective strategies for gender pre-selection were developed on these assumptions and claims of other philosophers. Although ANTON VAN LEEUWEHOEK (1677) described spermatozoa for the first time using an improved microscope, real scientific approaches of gender pre-selection were first undertaken in the 20th century with the development of more sophisticated instruments (HUNTER, 1995).

Natural mechanism of sex determination Nature has developed several systems to maintain a balanced distribution of genetic information including sex related mechanisms within the population. Major impacts on the development of such systems are related to environmental dependence of a species.

For example, in many reptiles, temperature depending enzymes regulate the sex of offspring (DORIZZI et al., 1996; GABRIEL et al., 2001; PIEAU et al., 2001).

However, in some reptile species, sex is determined chromosomally depending on the combination of sex chromosomes (ZW vs. ZZ) as found in lizards and turtles (CORIAT et al., 1994).

In mammals and avian species, primary regulation of sex determination depends on chromosomal information only. Whereas in the latter female gametes are heterogametic, mammalian spermatozoa carry either a Y- or X-chromosome (DAVIS, 1981; JACOBS and STRONG, 1959; MCLAREN and MONK, 1981). The Ychromosome carries the genetic information for a testis-determining factor (TDF) that initiates the formation of testicular material in the primitive genital ridge. Secondarily, the morphological and functional development of the male genital apparatus is mainly hormonal dependent and suppresses in parallel development of the female genital tract.

A precise localization of the TDF region and the genes that are included in sex differentiation were investigated comparatively between XY and XX males. DNA of these individuals includes different amount of Y-chromosome and their analysis allowed to map the short arm of Y-chromosome (MÜLLER et al., 1986). Further analysis of this region reveled that more genes are involved in sex differentiation.

Cloning and screening of this region first discovered the zinc-finger gene on the Ychromosome (ZFY) believed to be the testis determination factor (TDF; PAGE et al., 1987). However, evidence was provided that ZFY cannot be the TDF, because some of XX males were ZFY negative (PALMER et al., 1989). Another candidate for TDF is Arch. Tierz. 49 (2006) 1 the "sex region on the Y-chromosome" (SRY), which was discovered shortly after ZFY. Expression studies, mutational analysis of SRY in XY females, production of transgenic mice, and biochemical analysis provided further evidence that SRY is TDF (HARLEY et al., 1992; JAGER et al., 1990; KOOPMAN et al., 1990; KOOPMAN et al., 1991).

Techniques to identify sex-related characteristics of spermatozoa The most effective way to influence sex ratios in offspring is to determine the sex before fertilization and therefore separate populations of X- and Y-chromosome bearing spermatozoa. Several techniques based on principals such as velocity (BEAL et al., 1984; BEERNINK and ERICSSON, 1982; DMOWSKI et al., 1979; ERICSSON et al., 1973; ZAVOS, 1985), density (BHATTACHARYA, 1958;

BHATTACHARYA, 1962; BHATTACHARYA et al., 1966; FLAHERTY et al., 1997; KANEKO et al., 1983; KOBAYASHI et al., 2004; LOPEZ et al., 1993;

PYRZAK, 1994; QUINLIVAN et al., 1982; ROHDE et al., 1975; ROSS et al., 1975;

SCHILLING and THORMAEHLEN, 1977; SHASTRY et al., 1977; VIDAL et al., 1993; WANG et al., 1994b; WANG et al., 1994a), electric surface charge (BLOTTNER et al., 1994; ENGELMANN et al., 1988; MANGER et al., 1997;

SEVINC, 1968; SHIRAI et al., 1974; SHISHITO et al., 1974; UWLAND and WILLEMS, 1975), and immunologically relevant structures (ALI et al., 1990;

BENNETT and BOYSE, 1973; BLECHER et al., 1999; ERICKSON et al., 1981;

HANCOCK, 1978; HENDRIKSEN et al., 1993; PINKEL et al., 1985; SILLS et al.,

1998) have been developed and tested. None of these methods were able to produce statistically significant separation of fertile sperm populations, or were not repeatable.

The only method known so far uses the relative difference in DNA content of X- and Y- chromosome bearing spermatozoa. Research on DNA indicated by mid of the last century that its amount differs between the sex chromosomes. It led to the idea to identify spermatozoa on this basis. MORUZZI (1979) showed that the X-chromosome carries more DNA than the Y-chromosome, and that sperm autosomes have identical DNA content.

Development of flowcytometrical sperm sorting At the same time, flowcytometry was developed (SPRENGER et al., 1971) and GLEDHILL et al. (1976) reported about first experiments of flowcytometrical sperm analysis. Unfortunately, these experiments failed, until the problem of flat cell orientation was first solved for analysis of chicken erythrocytes, employing two sheath-liquid streams (FULWYLER, 1977). The adaptation of the injection tube to wedge shape and inclusion of a second light detector were necessary to gain higher resolution for analysis of flat cells (DEAN et al., 1978; STOVEL et al., 1978), Accordingly, PINKEL et al. (1982) modified their system to oriented spermatozoa in front of the laser beam. These improvements of the flowcytometer were the prerequisites to detect differences in DNA content of X- and Y- chromosome bearing spermatozoa, first reported by GARNER et al. (1983). In their initial projects, spermatozoa had to be fixed in ethanol for flowcytometrical analysis after being labelled with the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI) in order to achieve two different peaks representing X- and Y- sperm populations and to quantify the difference in DNA content between spermatozoa for bull (3.8%), boar (3.7%), ram (4.1%) and rabbit (3.9%). Due to the labeling process, spermatozoa had no fertilizing KLINC; RATH: Application of flowcytometrically sexed spermatozoa in different farm animal species abilities after sorting, but the experiment approved the differentiation of X- and Ychromosome bearing spermatozoa, opening a new field in biotechnology.

Several further modifications on the flowcytometer and in sample preparation were necessary to obtain higher resolution. JOHNSON and PINKEL (1986) modified a Coulter EPICS V flowcytometer, adding a second fluorescence detector at 0° and developed a beveled tip for the sample injection tube. Ethanol was still used for the fixation of the spermatozoa, but labeling of spermatozoa was performed now with Hoechst 33342, allowing specific and relatively uniform staining of the DNA (JOHNSON et al., 1987b); JOHNSON et al., 1987a). Despite evidence of its detrimental effect on cell growth (ERBA et al., 1988), Hoechst 33342 was chosen as the least toxic from all DNA fluorescent stains that have potentials to penetrate the plasma membrane of living cells (JOHNSON et al., 1987b). The stain selectively binds to A-T rich regions of DNA and enables the detection of small differences in DNA content. Based on the investigation of offspring produced from flowcytometrically sorted spermatozoa, no mutagenic or teratogenic effects of stain and/or sorting procedure were found so far (PARRILLA et al., 2004; TUBMAN et al., 2004).

The sorting process with a modified standard flowcytometer was relatively slow and allowed separation of about 55 sperm heads/second. In 1988 first evidence was seen that flowcytometrically sorted spermatozoa from domestic animals were able to decondense and form a pronucleus after sperm injection into a hamster oocyte (JOHNSON and CLARKE, 1988). Birth of the first animals inseminated with now viable sorted spermatozoa were reported in the same year (MORRELL et al., 1988). A year later surgical inseminations with sorted spermatozoa into the uterus of does resulted in the birth of offspring with significant shift in sex ratio. Among 37 offspring the proper sex was observed in 94% and 81% of animals after insemination with Xand Y-chromosome bearing spermatozoa, respectively (JOHNSON et al., 1989).

Sperm sorting with a flowcytometer requires several technical modifications of a standard apparatus to measure DNA difference in X- and Y-bearing spermatozoa. The flowcytometer has to be equipped with a 5W UV-light, water-cooled argon laser. This may chance now, as the first solid UV-lasers appeared on the market. Besides longer lifetime their signal can be pulsed in order to minimize negative effects on sperm integrity. Until 1996 sperm sorters worked as 'standard-speed' systems, where the samples are sorted with not more than 0.84 kg/cm2 of pressure giving a sort rate of about 350,000 spermatozoa/h (JOHNSON et al., 1989). New high-speed cell sorters like the MoFlo SX (Dakocytomation, USA) operate at sample pressures up to 4,22 kg/cm2, allowing to identify 30,000 events/sec and producing up to 15 million sorted spermatozoa of high purity above 90% per hour. A major reason for this improvement is due to a modified orienting nozzle that improved sperm orientation by more than 70% (RENS et al., 1998). The nozzle was further refined by XY, Inc. (Fort Collins, Colorado) to incorporate an orientating ceramic nozzle tip (CytonozzleTM) and orientation of the sperm population reaches now 85%.

Preparation and handling of semen before and after sorting has a major impact on semen quality and thereby fertility. Possibly major sources for cell violation are the dye; laser light, electric field and mechanical forces by high liquid pressure (MAXWELL and JOHNSON, 1999). To minimize this damage, constant temperature, osmolarity of buffers, pH and sterility of staining sheath, collection and cryopreservation media are essential.

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