Economics of selecting for sex: the most important genetic trait

Over 20,000 calves have resulted from artificial insemination (AI) of cattle with sexed, frozen/thawed sperm in the course of experimentation in several countries, and from commercial sales in the United Kingdom. This technology likely will become commercially available in many countries within a few years. Accuracy of the process is about 90% for either sex, and resulting calves appear to be no different from non-sexed controls in birthweight, mortality, rate of gain, and incidence of abnormalities. The main determinants of the extent of use of sexed sperm will be pregnancy rate and cost. Fertility of low doses (1.5 x 10(6)-2 x 10(6)) of sexed, frozen sperm for AI of heifers usually has been in the range of 70-80% of unsexed sperm at normal doses (10 x 10(6)-20 x 10(6) sperm) in well managed herds; it has been lower in poorly managed herds, and likely will be lower with lactating dairy cows. It is expected that fertility of sexed sperm will increase significantly due to very recent improvements in the hydrodynamics of the sexing process and potential improvements in cryopreservation procedures. It is unclear how sexed sperm will be priced; the cost of sexed sperm for cattle will likely be more than double the cost of unsexed sperm in most markets. The economic benefit of using sexed sperm also will depend on the baseline fertility of the herd since at lower fertility, it takes more doses of semen per calf produced. It is noted that for a small percentage of elite cattle, the economics of using sexed sperm do not depend primarily on increased production or efficiency of producing meat or milk, but rather on factors such as scarcity, tradition, cattle show winnings, and biosecurity during herd expansion. Until sorting efficiencies improve and costs decline, sales likely will be limited primarily to these niche markets. With near normal fertility and a premium for sexing in the range of US$ 10 per insemination dose, sexed sperm likely would become economically and environmentally beneficial for many, if not most populations of cattle being bred by AI.     

Overview of sexing sperm

Hundreds of thousands of off springs have been born as a result of AI with sexed sperm. Although this technology has been used for many species, the overwhelming majority of pregnancies have been in cattle, nearly all as a result of sperm that were sexed and subsequently frozen. The technology for sexing sperm has not changed greatly in the past 7 years, but refinements have speeded up the process and reduced damage to sperm. The process of commercialization of sexed sperm has accelerated recently. However, this technology is characterized by high costs, complexity of implementation and lower pregnancy rates than with control sperm. Nevertheless, sexed, frozen bovine sperm are being produced commercially in many countries, although from a limited number of bulls. The main application of sexed sperm to date has been to breed dairy heifers to produce female calves. Because of the slow speed of sexing sperm, fewer sperm are used per insemination dose of sexed than conventional sperm, and pregnancy rates with this product are often only slightly decreased. Successful use of sexed sperm requires excellent management of cattle, careful handling of sperm and use of skilled inseminators. As costs decline, sexed sperm will be used increasingly for cattle breeding, horse breeding and niche applications in other species.     

Application of sexed semen technology to in vitro embryo production in cattle

Use of sexed semen in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of predetermined sex. For thousands of years, livestock owners have desired a methodology to predetermine the sex of offspring for their herds. The ability to sort individual sperm cells into viable X- and Y-chromosome-bearing fractions made producers' sex selection dreams reality in the 1990s and now semen can be sexed with greater than 90% accuracy with use of a flow cytometric cell sorter. Several concerns regarding the implementation of sexed semen technology include the apparent lower fertility of sorted sperm, the lower survival of sorted sperm after cryopreservation and the reduced number of sperm that could be separated in a specified time period. These issues are discussed in this review. There are also a number of issues that appear to influence the success rates of using sexed semen to produce bovine embryos in vitro. These issues include reductions in fertilization rates, lower cleavage rates, blastocyst rates and pregnancy rates, partial capacitation of the sperm, dilute sperm samples and sire variation. These subjects are also addressed in this paper. Finally, we will describe a recent field trial in which female Holstein embryos produced using the combined technologies of sex-selected semen and microfluidics were transferred either as single or bilateral twin embryos into beef cattle recipients, demonstrating these technologies' contributions to viable embryo production. The results indicate that large-scale transfer of in vitro produced, Holstein heifer embryos to beef recipients is a feasible production scheme.     

Preselection of sex of offspring in swine for production: current status of the process and its application

It is estimated that as many as 30,000 offspring, mostly cattle, have been produced in the past 5 years using AI or some other means of transport with spermatozoa sexed by flow cytometric sperm sorting and DNA as the marker of differentiation. It is well documented that the only marker in sperm that can be effectively used for the separation of X- and Y-chromosome bearing spermatozoa is DNA. The method, as it is currently used worldwide, is commonly known as the Beltsville Sperm Sexing Technology. The method is based on the separation of sperm using flow cytometric sorting to sort fluorescently (Hoechst 33342) labeled sperm based on their relative content of DNA within each population of X- and Y-spermatozoa. Currently, sperm can be produced routinely at a rate of 15 million X- and an equal number of Y-sperm per hour. The technology is being applied in livestock, laboratory animals, and zoo animals; and in humans with a success rate of 90-95% in shifting the sex ratio of offspring. Delivery of sexed sperm to the site of fertilization varies with species. Conventional AI, intrauterine insemination, intra-tubal insemination, IVF with embryo transfer and deep intrauterine insemination are effectively used to obtain pregnancies dependent on species. Although sperm of all species can be sorted with high purity, achieving pregnancies with the low numbers of sperm needed for commercial application remains particularly elusive in swine. Deep intrauterine insemination with 50-100 million sexed boar sperm per AI has given encouragement to the view that insemination with one-fiftieth of the standard insemination number will be sufficient to achieve pregnancies with sexed sperm when specialized catheters are used. Catheter design, volume of inseminate, number of sexed sperm are areas where further development is needed before routine inseminations with sexed sperm can be conducted in swine. Cryopreservation of sex-sorted sperm has been routinely applied in cattle. Although piglets have been born from frozen sex-sorted boar sperm, freezing and processing protocols in combination with sex-sorted sperm are not yet optimal for routine use. This review will discuss the most recent results and advances in sex-sorting swine sperm with emphasis on what developments must take place for the sexing technology to be applied in commercial practice.     

Applications of sexed semen in cattle production

Sexed semen will contribute to increased profitability of dairy and beef cattle production in a variety of ways. It could be used to produce offspring of the desired sex from a particular mating to take advantage of differences in value of males and females for specific marketing purposes. Commercial dairy farmers, those who produce and market milk, could use sexed semen to produce replacement daughters from genetically superior cows and beef crossbred sons from the remainder of their cow population. To increase the rate of response to selection, seedstock dairy cattle breeders could produce bulls for progeny testing from a smaller number of elite dams by using sexed semen to ensure that all of them produced a son. Using sexed semen could then reduce the cost of progeny testing those bulls, because fewer matings would be necessary to produce any required number of daughters. Commercial beef cattle farmers, producing animals for eventual slaughter, could use sexed semen to capitalize on the higher value of male than female offspring for meat production. They could also use sexed semen to produce specialized, genetically superior replacement heifers from as small a proportion of the herd as possible. This would allow the remainder of the herd to produce male calves from bulls or breeds with superior genetic merit for growth, feed conversion efficiency, and carcass merit. Single-sex, bred-heifer systems, in which each female is sold for slaughter soon after weaning her replacement daughter, would be possible with the use of X-chromosome-sorted semen. Use of sexed semen would make terminal crossbreeding systems more efficient and sustainable in beef cattle. Fewer females would be required to produce specialized maternal crossbred daughters, and more could be devoted to producing highly efficient, terminal crossbred sons.     

Pregnancy rates in cattle with cryopreserved sexed sperm: effects of sperm numbers per inseminate and site of sperm deposition

In six field trials, doses between 1.0 and 6.0 x 10(6) total sexed, frozen-thawed sperm were inseminated into the uterine body or bilaterally into the uterine horns of heifers and nursing Angus cows 12 or 24h after observed estrus. Except for one comparison in one trial in which uterine body insemination was slightly superior (P<0.05) to uterine horn insemination, there was no significant (P>0.1) difference between sites of semen deposition. Additionally, except for one small study with limited numbers, there was essentially no difference in pregnancy rates in the range between 1.5 and 6 x 10(6) sexed, frozen-thawed sperm per inseminate. Pregnancy rates with smaller doses of sexed sperm averaged about 75% of controls of 20 x 10(6) total frozen-thawed, unsexed sperm. While 1.0 x 10(6) sexed, frozen-thawed sperm per insemination dose resulted in decreased pregnancy rates compared to larger doses, the lesser fertility with sexed sperm could not be compensated by increasing sperm numbers in the range of 1.5-6 x 10(6) sperm per dose. Pregnancy rates with 2 x 10(6) sexed, frozen-thawed sperm per dose were not markedly less than control pregnancy rates with 20 x 10(6) frozen-thawed unsexed sperm/dose in well-managed herds.     

What affects fertility of sexed bull semen more, low sperm dosage or the sorting process?

Until now it has been unclear to what extent the reduced fertility with sexed semen in the dairy industry is caused by too few sperm per AI dose, or by the effect of flow cytometric sorting, which is the established procedure for sexing semen. Therefore, we evaluated the effects of low sperm numbers per dose with and without sorting on non-return rates after 56 days (NRR56); in addition, we evaluated the effects of bulls, in order to further optimize use of sexed semen.Based on results of using sexed semen from seven Holstein bulls, an overall numerical decline of 13.6% in NRR56 was observed (P < 0.05). About two-thirds of this decline (8.6%) was due to the low dose (P < 0.05), and a third (5.0%) due to the process of sorting (P < 0.05). The effect of low dosage and sorting differed among bulls. We observed a sex ratio of 91.6% females for sexed semen from the first 131 calves born.Currently the best way to increase fertility of sexed semen is by closely monitoring fertility so that the highest fertility bulls are used, and by improving farm animal management. However, to make substantial progress, more in depth studies are needed on the sexing technology, especially on aspects such as sorting procedures and sperm dosage.     

Pregnancy rates following AI with sexed semen in Mediterranean Italian buffalo heifers (Bubalus bubalis)

The use of sexed semen in farm animal production and genetic improvement has been shown to be feasible with variable degree of efficiency in a number of species, and proved to be economically viable in cattle. In the last two decades, various newly developed reproductive technologies applicable in buffaloes have mushroomed. Recently, following the birth of the first buffalo calves using AI with sexed semen, commercial interest to exploit sexing of semen in this species too is aroused. In order to verify the successful adoption of this technology in the buffalo, the present study on the use of sexed semen for AI was carried out and compared with conventional artificial insemination using nonsexed semen. A total of 379 buffalo heifers were used for synchronization of ovulation using the Presynch protocol in the South of Italy. Selected animals at the time of AI were randomly allocated to three different experiment groups: (1) 102 animals subjected to AI in the body of the uterus with sexed semen (SS body); (2) 104 animals subjected to AI in the horn of the uterus with sexed semen (SS horn); and (3) 106 animals subjected to AI in the body of the uterus with conventional nonsexed semen (NSS body). Semen of three buffalo bulls was sexed by a collaborating company and commercially distributed in 0.25 mL straws with a total of 2 million sexed spermatozoa. Pregnancy rates were first assessed at Day 28 following AI, and rechecked at Day 45 by ultrasound. Pregnancy rates were nonsignificantly different between animals inseminated with sexed or nonsexed semen: 80/206 (38.8%) and 40/106 (37.7%), respectively (P = 0.85). However, site of insemination of sexed semen affected pregnancy rate significantly as higher pregnancy rates were obtained when sexed semen was deposited into the body rather than the horn of the uterus: 46/101 (45.5%) and 34/105 (32.3%), respectively (P = 0.05). In conclusion, the use of sexed semen in buffalo heifers gave satisfactory and similar pregnancy rates when compared with conventional nonsexed semen. Deposition of sexed semen into the body of the uterus, however, increased pregnancy rates significantly.     

History of commercializing sexed semen for cattle

Although the basic principles controlling the sex of mammalian offspring have been known for a relatively long time, recent application of certain modern cellular methodologies has led to development of a flow cytometric system capable of differentiating and separating living X- and Y-chromosome-bearing sperm in amounts suitable for AI and therefore, commercialization of this sexing technology. After a very long history of unsuccessful attempts to differentiate between mammalian sperm that produce males from those that produce females, a breakthrough came in 1981 when it was demonstrated that precise DNA content could be measured. Although these initial measurements of DNA content killed the sperm in the process, they led to the ultimate development of a sperm sorting system that was capable, not only of differentiating between live X- and Y-sperm, but of sorting them into relatively pure X- and Y-sperm populations without obvious cellular damage. Initial efforts to predetermine the sex of mammalian offspring in 1989 required surgical insemination, but later enhancements provided sex-sorted sperm in quantities suitable for use with IVF. Subsequent advances in flow sorting provided minimal numbers of sperm sufficient for use in AI. It was not until the flow cytometric sorting system was improved greatly and successful cryopreservation of sex-sorted bull sperm was developed that efficacious approaches to commercialization of sexed semen could be implemented worldwide in cattle. A number of companies now offer sex-sorted bovine sperm. Innovative approaches by a diverse group of scientists along with advances in computer science, biophysics, cell biology, instrumentation, and applied reproductive physiology provided the basis for commercializing sexed semen in cattle.     

Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency

Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X-bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.     

Fertilization rates and in vitro embryo production using sexed or non-sexed semen selected with a silane-coated silica colloid or Percoll

The aim of this study was to evaluate sperm fertilization rates and in vitro embryo development rates for sexed and non-sexed semen selected using a silane-coated silica colloid method (Isolate) or Percoll. Frozen/thawed, sexed and unsexed semen samples from four Holstein bulls were randomly allocated to one of two different density gradient selection methods. Sperm quality (motility, concentration, morphology and membrane integrity) were evaluated and compared before and after sperm selection. Sperm motility and morphology improved (P < 0.005) after the sperm selection process with no differences between the two methods. For non-sexed semen, Percoll gradient increased the mean (± SEM) percentage of sperm recovered (57.3 ± 2.8) compared to Isolate (46.0 ± 1.8; P < 0.01). However, membrane integrity was higher after Isolate than Percoll (sexed semen: 41.0 ± 0.6 vs. 38.8 ± 0.8 and non-sexed semen 60.8 ± 1.6 vs. 58.8 ± 0.5; P < 0.05). The percentage of blastocysts produced was higher when either sexed or non-sexed semen was selected by Isolate (14.0 ± 1.0; 22.0 ± 1.1) than by Percoll (10.5 ± 1.5; 17.0 ± 2.1, respectively; P < 0.05). In summary, Isolate was a more effective method for the recovery of high quality sperm for in vitro fertilization embryo production.     

Issues affecting commercialization of sexed sperm

A decision tree for genetics or sperm-sexing entities considering sales of sexed sperm is discussed in terms of: (a) how best to avoid harm; (b) how best to do good; (c) needed synergy with other assisted reproductive technologies; (d) constraints on biotechnology; and (e) costs with current and likely technologies versus potential benefits to producers. The sexed-sperm industry might wish to take a pro-active stance on societal issues potentially affecting use of sexed sperm. For most sales in animal agriculture, cost of added value must be < 50% of benefit. Cost is less important for emotionally-driven uses with horses and human beings. Current procedures for flow-sorting allow most sperm to retain their fertilizing potential. Added cost to produce and package 2 x 10(6) sperm is estimated at US $30 to US $46 with flow sorted sperm. Estimating cost of any alternative technology is premature. For IVF/embryo transfer (ET), cost and numbers of flow-sorted sexed sperm are appropriate for commercial use. For use in low-dose AI, however, added cost to supply one insemination dose must be near US $12. Flow-sorting instruments with higher throughput and lower purchase and operating costs are obligatory for economic application in most AI situations. Developers of antibody-based separations also will face issues of retention of fertilizing potential while minimizing cell loss, separation of living from dead sperm concurrent with sperm sexing, output, and cost. To benefit producers and consumers in a changing world, genetics and sperm-sexing companies will have to collaborate and interface to provide funding for needed research and development and to recover these costs, using mechanisms not yet obvious.     

Production of piglets with sexed semen employing a non-surgical insemination technique

The aim of the present study was to ascertain whether multiparous sows could successfully be inseminated with sexed semen non-surgically. Spermatozoa were stained with Hoechst 33342 and separated flowcytometrically in X- and Y-chromosome bearing sperm populations employing the Beltsville Sperm Sexing Technology (BSST). After weaning, estrus was induced in sows with PMSG and hCG. Animals were inseminated once per estrus non-surgically with a specially designed catheter into the tip of the uterine horn, employing 50x10(6) of either sexed or non-sexed spermatozoa diluted in 2 ml Androhep. Pregnant sows were allowed to go to term. Mean pregnancy rate from inseminations with unsexed spermatozoa was 54.5% whereas inseminations with sexed spermatozoa resulted in 33.3% pregnant sows. All but one piglet born after insemination with sexed semen were of the predicted sex. The sex of those piglets born after inseminations with non-sexed spermatozoa was 61.1% for male and 38.9% for female sex. It is concluded that non-surgically inseminations with flowcytometrically sexed spermatozoa can be conducted successfully.     

Adjusting the number of spermatozoa in mini-straws by determination of the operative volume of bovine semen

The importance of the number of sperm per insemination on fertility has been well demonstrated in cattle. This number is usually calculated from the concentration in the extended semen and a theoretical value for the operative volume of semen delivered during insemination. The objective of this experiment was to investigate the usefulness of the measurement of the delivered volume of semen when estimating sperm numbers from frozen-thawed mini-straws, by comparing the results obtained with an analytical balance to the theoretical volume. The density of semen extended with Biociphos Plus and Triladyl was determined to be 1.033 g/ml using the gamma sphere method. This value was used to convert semen weight into operative volume. The effect of semen temperature at the time of weighing (37 degrees C versus 20 degrees C) was investigated on six semen batches, two technicians measuring the operative volume of 50 straws for each combination of temperature and semen batch (a total of 1200 weighings). The temperature effect was found to be insignificant, which allowed warm semen to be weighed before motility was assessed during routine quality control. The operative volume was then measured in straws routinely produced at 17 bovine Al centers (12-105 semen batches per center, mostly three straws from each batch, a total of 1912 measurements). The observed volumes were normally distributed around 198.7 microl, 98% measuring between 180 and 210 microl. The operative volume was significantly different among centers (from 192 to 205 microl, P < 0.0001) and among batches within centers (P < 0.0001). The S.D. among straws within batches was 3.4 microl. Some centers showed high variability in straw volume whereas others were more consistent. Determination of the operative volume of frozen-thawed mini-straws by weighing the delivered contents is an accurate method for estimation of the number of sperm per dose.     

Embryo production in superovulated Angus cows inseminated four times with sexed-sorted or conventional, frozen-thawed semen

This study tested the hypothesis that four inseminations of commercially frozen sexed semen (≥2.1 × 10⁶ sperm per 0.25-mL straw) in superstimulated embryo donors would yield a percentage and quantity of transferable embryos similar to that achieved with conventional frozen semen. Bos taurus, angus cows (n = 32), stratified by age and body condition, were randomly allocated to receive four inseminations of frozen-thawed semen, either conventional semen (≥15 × 10⁶ sperm/straw; Conventional) or sexed semen (≥2.1 × 10⁶ sperm/straw; Sexed) from one of two AI sires. From 10 to 13 d after estrus, follicle-stimulating hormone (FSH) was given twice-daily, with prostaglandin F_2α given twice on the last day. Cows were inseminated once (1×) at first detected estrus and twice (2×) and once (1×) at 12 and 24 h later, respectively, with nonsurgical embryo recovery 7 d after first detected estrus. The study was repeated 30 d later (switch-back experimental design). The total number of ova per flush was similar between Conventional and Sexed treatments (10.9 ± 1.8 vs. 10.5 ± 1.6), but the number of Grade 1 embryos was greater (P < 0.01) for Conventional (4.3 ± 0.8 vs. 2.3 ± 0.7). Conversely, the mean number of unfertilized ova was greater (P < 0.05) for Sexed (5.6 ± 1.0 vs. 3.0 ± 1.2). There was no significant difference between treatments for numbers of degenerate, Grades 2 or 3, and transferable embryos and no significant differences between bulls in percentage of transferable embryos (44.4% and 46.7%). However, fertilization rates and percentage of transferable embryos were affected (P < 0.05) by period and donor. In conclusion, superstimulated donor cows inseminated four times had fewer Grade 1 embryos and more unfertilized ova with sexed versus conventional semen.     

Sperm sexing technology-the transition to commercial application. An introduction to the symposium "update on sexing mammalian sperm"

Over the past decade, procedures for sexing mammalian sperm by flow cytometry/cell sorting have been refined sufficiently for large-scale commercial application in cattle; millions of doses of sexed sperm are sold each year for artificial insemination. Furthermore, more than a hundred babies are born annually from use of sexed human sperm via artificial insemination or in vitro fertilization, and to a lesser extent by injection of single sperm into oocytes. Semen sexing technology is also being applied for various objectives in a number of other species. Accuracy of sexing is around 90% in most species. However, this technology is still rather expensive, and fertility of sexed sperm is lower than unsexed controls in most instances. Speakers in this symposium, “Update on sexing mammalian sperm,” held in San Diego, California on 3 January 2009, presented the latest research on sexing sperm, including documentation of success rates. The written versions were peer-reviewed and follow in this issue of Theriogenology.     

In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up

The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.     

Evaluating the success of sex-sorted semen in US dairy herds from on farm records

These data summarize on-farm records of dairy herds (n = 211) using sexed semen. Sexed semen was predominantly used at first and second service in virgin heifers, which is reflected in younger ages at AI and at calving. Conception rates at first service averaged 47% for Holstein heifers and 53% for Jersey heifers, which were ∼80% of that achieved with conventional semen. Analysis of inter-estrus intervals provides no evidence that cycle lengths are extended by use of sexed semen. Among singleton births, 89% were reported as female offspring and this rises to 90% for gestation lengths within a normal 265-295 d range. Age at calving appeared to interact with calf sex and semen type to influence the incidence of stillbirths. Semen type had no effect on the incidence of stillbirths among heifers delivering female calves. However, the incidence of stillbirths among heifers delivering male calves was greater for those conceived from sexed semen and was only partially explained by age at calving. Because the incidence of male calves from sexed semen is only 10%, the total incidence of stillbirths was not affected by semen type. In conclusion, failure to differentiate sexed from conventional semen in data recording and preferential bias in use of sexed semen in younger, more fertile females makes legitimate comparisons of sexed and conventional semen in the commercial setting difficult. When used in Holstein heifers, the average first service conception rate achieved with sex-sorted semen was 47%, which appeared to ∼80% of that achieved with conventional semen in the same herds. The percentage of female calves (89%) was consistent with expectations. After adjusting for age at calving, sexed semen had no affect on the total incidence of stillbirths, however the source for an apparent increased incidence of stillbirth among male calves born from X-sorted sperm populations requires further investigation.     

Flow cytometric sexing of mammalian sperm

This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species.     

In vitro fertilization with flow-cytometrically-sorted bovine sperm

An attractive feature of IVF is that fewer sexed sperm are needed than for artificial insemination. However, sperm sexed by flow cytometry/cell sorting are probably pre-capacitated, necessitating modifications to standard IVF systems for optimal success. With current procedures, the percentages of oocytes fertilized with sorted and unsorted frozen bovine sperm are similar, and events during the first cell cycle are timed similarly for sorted and unsorted sperm. However, in most cases, blastocyst production with sorted sperm was approximately 70% of controls produced with unsorted sperm. In some early studies, there appeared to be an unexplained delay of about half a day in blastocyst development. Nevertheless, some dozens of apparently normal calves, pre-sexed with 90% accuracy, have resulted from frozen embryos produced via IVF with sexed sperm. IVF also has proven useful as a bioassay for improving sperm-sorting procedures such as determining potential detrimental effects of laser power. It is likely that use of IVF in cattle breeding programs will increase considerably when sexed, frozen sperm become commercially available.