Sexing Bull Sperm

Sexing Bull Sperm

The sex of calves now can be predetermined with 85 - 95% accuracy [1,2]. This is accomplished by sorting sperm with a flow cytometer/cell sorter. This technique, which was developed for live sperm by Dr. Larry A. Johnson at the USDA Beltsville Agricultural Research Center, is patented [3-6] and exclusively licensed worldwide for non-human mammals to XY, Inc., a private company. This company has invested heavily into advancing this technology to the point that it has become a commercial reality [7,8].

Sperm Sexing Process

Semen can be sexed because X-sperm, which produce heifers, contain 3.8% more DNA than Y-sperm, which produce bull calves [5]. Freshly collected sperm are stained with a specific bisbenzimidazole DNA-binding dye, Hoechst 33342, for 1 h [9]. The stained sperm fluoresce bright blue when exposed to a laser beam of short wavelength light. The stained X-sperm emit a brighter fluorescence than Y-sperm because of the greater DNA content. This difference in emitted fluorescence can be measured by a photomultiplier tube (PMT) and integrated using a powerful computer so that DNA content for most, but not all sperm can be accurately determined as they pass the PMT in a stream of fluid. As the fluid stream containing the sperm exits the nozzle of the sorter, it is vibrated at a high frequency causing individual droplets to form at a rate of about 90,000/sec [9]. Although not all droplets contain sperm, those that do are given a positive or negative charge, depending on the DNA content information that was provided by detector. Sperm must be oriented properly to measure DNA content accurately [5]. No charge is applied to droplets containing more than one sperm, dead sperm as determined by uptake of a vital dye, or those sperm where DNA content could not be measured accurately, thereby simply allowing these cells to be disposed of as waste. Charged droplets containing X-sperm, which had been negatively- or positively charged according to sperm DNA content, are deflected by an oppositely charged plate thereby directing the sperm into a collection vessel. Droplets containing Y-sperm are simultaneously directed to a different collection vessel by applying an opposite charge to those drops so that the sperm are deflected toward an opposing-charged plate. Thus, the streams of droplets containing the X-sperm, the Y-sperm or no sperm or too many sperm are collected into three separate vessels. This process allows sexing and collection of about 40% of the sperm going through the sorter at a speed of approximately 100 km/h. Thus, at an event rate of 20,000 total sperm/sec, nearly 4,000 live sperm/sec of each sex can be sorted simultaneously [9]. The current system can produce approximately 10 to 13 x 106 live sperm/h of each sex with 85 - 95% accuracy.

Low-Dose Insemination

The sorting process dilutes sperm, so they need to be re-concentrated by centrifugation prior to packaging in 0.25 ml French straws at doses of 1 to 6 x 106 sperm/straw. Conventional artificial insemination (AI) procedures utilize around 20 x 106 sperm/straw, so the insemination dose of sex-sorted sperm is about 1/20 to normal, frozen-semen AI dose. Even at the achieved superior sort rates, it is necessary to use 1/3 fewer sperm per insemination than used conventionally to have a commercial product [10]. In recent work in Holland, normal non-return rates were achieved with less than 2 x 106 sperm when semen from highly fertile bulls was used [11]. Utilization of only the top 20% of fertile bulls would certainly expedite implementation of sexed sperm. Low-dose insemination allows many more heifers to be bred with sex-sorted sperm than with current AI practices. Under the current system for sexing, bovine sperm are cryopreserved so that they can be efficiently used for AI of estrus-synchronized heifers. The inherently higher fertility of heifers makes them more suitable than cows for the current sexing technology. Preliminary results using low-doses of sex-sorted sperm for breeding lactating cows have been less than satisfactory [1].

Estrus Synchronization

Optimal results can be obtained only if the heifers to be inseminated are properly managed [1]. Several successful regimens have been used to synchronize estrus for sexed sperm including 1) 500 mg of melengestrol acetate (MGA) fed daily in grain for 14 d followed by an IM injection of 25 mg prostaglandin F2α 17, 18 or 19 d after the last day of feeding MGA;  2) a single injection IM of 25 mg prostaglandin F2α;  3) 20 or 25 mg prostaglandin F2α injected IM at 12-d intervals;  and 4) 50 or 100 μg of  GnRH injected IM followed by 25 mg of prostaglandin F2α 7d later.

Insemination

Heifers were inseminated after 16:00 h so that the insemination occurred approximately ½ or 1 d following the onset of estrus [1], with no significant difference in pregnancy rates with sexed sperm between these times (45 and 49%, respectively). Straws containing the sex-sorted sperm were thawed for 20 to 30 sec in a 34 to 37°C water bath before being immediately inseminated at either of two sites, into the lumen of the uterine body, as is done for conventional AI, or deep in the uterine horn [1]. Between May 1998 and July 1999, more than 1,000 heifers were bred by 7 inseminators using sex-sorted, cryopreserved sperm from 22 bulls of unknown fertility representing various dairy and beef breeds [1].

Pregnancy Rates

later trials of this group, the pregnancy rates for sexed, cryopreserved sperm sometimes were 90% of small, non-significant differences in pregnancy rates were noted between 1.0 to 1.5 x 106 vs. 3.0 x 106 sexed, cryopreserved sperm for the 1,000 inseminations described above [1] (Table 1). Furthermore in some of the controls that had 7 to 20 times more sperm/insemination dose [1]. In some early trials, pregnancy rates for controls were much higher than for sexed sperm. Only in one trial did inseminations into the uterine horn result in higher pregnancy rates than when the sperm were placed in the uterine body as is done for conventional AI [1]. No differences were noted among inseminators. Some bull differences were suggested, but numbers of inseminations per bull were inadequate to draw any firm conclusion.

Calving Results

Not all animals were followed to calving due to the need for collaborators to market the animals as “springer” heifers. Several hundred heifers have been followed to term and many more are currently gestating. In those animals that were followed to term, there did not appear to be any excess embryonic deaths between 1 and 2 months of gestation, and very few abortions occurred between 2 months gestation and term [1]. Rigorous epidemiological studies, however, need to be done to confirm that calves from sexed sperm are completely normal. Recently, three heifers that were produced from sexed sperm calved as a result of being themselves inseminated with sexed sperm. This second generation of calves from sexed, cryopreserved sperm demonstrates the feasibility of using an all heifer production system, whereby heifers are bred with sexed sperm to produce their own replacements [12].

In Vitro Fertilization (IVF)

Although the first sexed-selected calves were born from embryos derived from IVF with sex-sorted sperm [13], most of the recent work has been done using AI [1,14]. Large-scale trials are now underway to examine the normalcy of calves born from IVF-produced embryos.

Summary

Commercialization of sexed sperm for AI in heifers is imminent.