Sexed semen: Challenges and Opportunities for Indian Dairy Industry

Sexed semen: Challenges and Opportunities for Indian Dairy Industry

 

In India, the demand for female calf is gaining much attention day by day especially in the dairy sectors. Drop in crop yield by disaster and also decreasing land productivity one hand and constant increase in the requirement and consumption of good quality of dairy product on the other hand due to raise in purchasing power of the average population, urbanization and increased population. There is an immediate need to expand the genetic merit of dairy animals. In 20^th livestock census India’s population reached 536.76 million (showed an increase of 4.6% over the 19^th Livestock Census). In order to accommodate food demand associated with this population growth it will be needed to produce pre-sexed livestock by sperm or embryo sexing; which offer an encouraging breeding strategy to meet the expanded demand for food production and nutritional security. Application of sexed bovine sperm by using Artificial Insemination is effective in modifying the sex ratio and promptly expanding dairy herds transmit high genetic value animals. It also increases the rate of genetic progress, especially in combination with genomic selection of sires and smooth culling decisions. It also has the potential to help in herd management and cut down the incidence of dystocia by avoiding male calves.

Sexing of semen is based on the principle of difference in DNA content between X and Y spermatozoa. X spermatozoa hold more DNA. With the help of sexed semen technology sorting of X and Y bearing chromosome to produce progeny of a desired sex with 80 to 90% accuracy. But, this technology holds up by many factors like high costs, complexity of operation and lower conception rates than with traditional semen. Despite the drawback, sexed semen will contribute to increased profitability of dairy cattle production.

What is sexed semen?

Semen contains an X or Y bearing sperms to produce a progeny of a desired sex either female or male with having 80 to 90% accuracy. It’s coming into existence in 1981 when it was proven that the precise DNA content could be quantified. This technology was developed by the United States Department of Agriculture (USDA) researchers in Livermore, California and Beltsville, Maryland and patented as “Beltsville sperm sexing technology. The introduction of sexed semen was done by US commercial organizations in 2005 (DeJarnette et al., 2009). Sexed semen is produced in India for the first time under the brand name “Sexcel” in 2017 by ABS India using their proprietary IntelliGen technology. First male calf named Shreyas was born in India on 1st January, 2011 by using sexed semen. Sexed semen is being used in about 9 % of all reported breeding in the year 2017 on the globe and is showing a double-digit growth in usage year on year (Sharma et al., 2019).

What are the methods of sperm sexing?

Different  methods are used for sperm sexing like Percoll density gradient, Albumin gradient, Free-flow electrophoresis, Identification of  HY antigen and Flow  cytometry for sperm sexing. The sex sorting process by flow cytometry is the most efficient method to separate X from Y spermatozoa in a large scale (Fig 1 & 2) (Rath et al., 2013). In this technique separation of female sperm cells from male sperm cells is done without any change in the sperm morphology (Table 1). Sperm sexing measures variation in DNA%. X bearing chromosome contains about 4 % more DNA than Y bearing chromosome (Johnson, 2000).

Parameter	Difference
DNA content	Less in Y sperms
Size	X sperm is larger
Motility	Y sperm is faster
Surface charge	X sperm is negative
Cell surface antigen	H-Y antigen on Y sperm
Sperm surface	Specific protein
F- body	Long arm on Y- chromosome

Table 1.  Differences in X and Y Spermatozoa (Johnson,1995)

How to measure DNA?

The DNA content of the sperm is measure by fluorescent dye Hoechst -33342 (a DNA binding fluorochrome [2-(4-ethoxyphenyl)-5-(4-methyl-1- piperazinyl)-2, 5-bis-1H-benzimadazole-trihydrochloride). This dye penetrates the sperm membrane and then bind, the site where A-T region of nucleic acids are present.  X-sperm binds more dye to their DNA than Y-sperm. When the  low wavelength laser beam are exposed to these sperm cell then X-sperm cell gives off more fluorescence because it contain more 3-4% more DNA content as compared to Y-sperm. The charge is applied to the droplet having desired cells and these are subsequently deflected into the required population of the sexed cells resulting in high purity and viability.

Fig 1.Flow chart showing step wise procedure of sex sorting using flow cytometry

Fig 2. Schematic Diagram of Sex Sorting using Flow Cytometry.(Source: Dairy Knowledge portal, NDDB)

 

Methods of sperm sexing

 

There are 60 chromosomes in each somatic cell of domestic cattle. The gametes contain half that number of total chromosome. Half of the gametes have haploid sperm that produce females carry 29 autosomes plus the X-chromosome. Similarly rest half haploid Y-chromosome bearing sperm have the same 29 autosomes plus the male determining Y-chromosome. It was observed that sperms bearing Xchromosomes contain approximately 4.2% more DNA than sperm containing Ychromosomes in domestic cattle (Moruzzi, 1979). Besides difference in sex chromosome in gametes, many other differences were discovered during last four decades of 20th century. Size of spermatozoa with X chromosome is larger than the spermatozoa contains Y chromosome (Cui and Matthews, 1993 and Cui, 1997). Comparatively higher motility reported in spermatozoa containing Y chromosome than those bearing X chromosome (Shettles, 1960). There are difference in surface charges in spermatozoa as X chromosome bearing spermatozoa has a negative charge while Y chromosome bearing spermatozoa has a positive charge (Kiddy and Hafs, 1971). Difference in cell surface antigens among X and Y bearing chromosomes was also reported by Hoppe and Koo (1984). On the basis of these potential differences, various methods were proposed for sorting of spermatozoa.

Gradient swim down procedure of sorting of spermatozoa:

The basic principle of sorting through this method is due to difference in ability of X and Y bearing spermatozoa to swim down in a gradient solution. Y chromosome bearing spermatozoa have high motility and exhibiting greater downward swimming velocity due to its small size than X chromosome bearing spermatozoa. Hence the isolation of fraction of semen from specific part of albumin gradient shows higher proportion of X or Y spermatozoa at different gradients but the success rate is reported to be around 75% (Beernink et al., 1993).

Sperm sorting by swim-up procedure:

Due to small size, Y chromosome bearing spermatozoa swim faster than X chromosome bearing spermatozoa and this difference was utilized by many researchers for sperm sorting (Van Munster et al., 1999 and Ollero et al., 2000). The success rate was recorded up to 81% by this method (Check et al., 1989). Sperm sorting by identification of H-Y antigen: Using specific antibody against surface protein of Y chromosome bearing spermatozoa (against H-Y antigen) is also an option for sperm sorting through affinity chromatography or magnetic bead. This sperm sorting is applied at large scale with efficacy reported to be more than 90% by many scientists (Hoppe and Koo, 1984; Hendriksen et al., 1996; Hendriksen, 1999 and Blecher et al., 1999).

Sperm sorting by free flow electrophoresis :

Difference in surface charges in spermatozoa (as X chromosome bearing spermatozoa has a negative charge while Y chromosome bearing spermatozoa has a positive charge) is utilized for sperm sorting by electric field separation (Mohri et al., 1986 and Kaneko et al., 1984).

 

Sperm sorting by percoll density gradient method :

Sedimentation density of X chromosome bearing spermatozoa is higher and settles in the bottom of column while the Y chromosome bearing spermatozoa remain in high proportion at the top of column during sperm sorting. The success rate is reported to be from 86% to 94% (Lizuka et al., 1987; Van Kooij and Van Oost, 1992).

Sperm sorting by flow cytometry :

Flow cytometry is a method used to sort sperm and adaptations of this technique opens new opportunities in sperm sorting. Flow cytometry-based sperm sorting uses fluorescent dyes that stain DNA (Caroppo, 2013 and Ribeiro et al., 2013). This technique is able to determine the sex of future progeny by measuring DNA content of individual sperm cells as if they contain the larger X chromosome (giving rise to a female offspring) or smaller Y chromosome (leading to male progeny). It then allows separation of X and Y sperm (Johnson et al., 1989). The so-called Beltsfield sperm sexing technology was developed by USDA in conjunction with Lawrence Livermore National Laboratories, relying on the DNA difference between the X and Y chromosomes (Garner and Seidel, 2008). In this method, first, sperm are stained with a non-toxic, DNA-binding dye (Hoechst 33342) and pumped in a stream in front of UV laser beam having wavelength of 351 – 364 nm and the bright blue fluorescence emitted is detected and analyzed (Johnson and Welch, 1999). This stream is broken by crystal vibrator into individual droplets for facilitating analysis of individual spermatozoa. Bright fluorescence emitted by illuminated spermatozoa which is measured rapidly by a photo-multiplier tube as the sperm flow past in single file (Garner and Seidel, 2008). To ensure adequate illumination, the sperm stream is oriented at the appropriate angle for accurate measurement of a 4% difference in fluorescence (Sharpe and Evans, 2009). The relative fluorescence of X and Y chromosome bearing sperm population is analyzed by high speed computer, which are then sorted by DNA content by introducing opposite charges on droplets containing X chromosome bearing sperm than Y chromosome bearing sperm (Seidel Jr, 2007). These droplets falls on previously charged deflector plates thus separated into two streams and then collected separately. The separation of streams of X and Y chromosome bearing droplets are done by using electrostatic deflection and collected separately for further processing (Seidel Jr and Garner, 2002). A third stream of uncharged droplets passes through as waste and is discarded (Seidel Jr, 2007). Many preliminary results have since been confirmed by a number of larger studies that have demonstrated CR with frozenthawed sexed semen (2×106 sperm/ straw) that were ~70% to 80% of the conception rate achieved with frozen-thawed conventional semen (15 to 20×106 sperm/ straw) in both virgin heifers and lactating cows (DeJarnette et al., 2010; Norman et al., 2010; DeJarnette et al., 2011 and Healy et al., 2013). This method of sex specific spermatozoa sorting is the most popular and consistently proven to be effective among various methods.

 

Scope of semen sexing technology in India

In Indian context, the sexed semen has following potential applications

• To realize the increasing demand of milk production: The projected demand of milk by 2020 is estimated to be 191.3 MT. To meet the increasing demands, it is necessary to substantially increase the number of elite female which can achieved by shifting the sex ratio towards females. The sexed semen bearing X chromosome could be used in elite cows to produce superior high yielding cows at a faster rate than the conventional unsexed semen.
• Production of superior breeding bulls: In our country there is shortage of superior breeding bulls. The projected additional frozen semen doses required per year (millions) is 48, 9.6 and 52.8 for indigenous, crossbred and buffalo respectively (Mohanty et al., 2013). By introducing sexed male sperm, superior bulls could be produced from the limited number of elite cows available. But, superior male can be produced by sex sorted spermatozoa from superior dam, which will be a great boost for semen station which is the need of the hour for increasing the frozen semen productivity in the country.
• Progeny testing: Sexed semen technology ensure required number of progenies per bull under progeny testing programme, thereby increases the accuracy of bull testing. The sexed female sperms could be used in test mating so as to ensure the production of required number of daughters in shortest time, thus increasing the genetic gain.
• Reducing the number of unproductive young bulls: The percentage of total males used for breeding and allied activities are 36.6, 64.8 and 29.56 in case of crossbred, indigenous and buffalo respectively (Mohanty et al., 2013). Thus there is large number of unproductive young bulls. Since, cattle slaughter is banned nearly all over India, male calves produced through artificial insemination are of little use in terms of either future breeding bulls or bullocks in the agricultural fields as a source of farm power. The use of sexed semen can solve the problem of production of unwanted male progenies.
• Replacement and expansion of herds: Sexed semen is an excellent way to expand the dairy herd without spending large amount on replacement and virtually a breeder need not to buy new heifers. The benefits of sexed semen can be further amplified by coupling it with assisted reproductive techniques like multiple ovulation embryo transfer technology, In-vitro fertilization, Gamete intra-fallopian transfer and Sperm intra-fallopian transfer. These technologies may help to overcome the lower fertility problem.

 

Importance of sperm sexing

• Unwanted male calves can be minimized which help the farmers to save resources wastage on management of male animals.
• Helpful in genetic improvement in case of selection of best female for different traits.
• Sexed semen can be used for herd replacements and new heifers for herd expansion at a faster rate from within the herd, thereby, reduce Biosecurity risks associated with purchasing a new animal from outside the herd.
• For progeny testing program, it ensures required number of daughters under short time, thus it increase genetic gain.
• During the sorting process, dead sperms are removed, which shows the existence of only viable sperms.
• Reduce the chance of dystocia cases by avoiding production of male calves by use of sexed semen
• Also helpful in the production of superior breeding bulls as India has limited elite cattle and buffalo bulls.

AI strategies with sex sorted sperm

Even though the significant advances in sex-sorting sperm using flow cytometry in cattle, lower pregnancy per AI (P/AI) and reduced In vivo embryo production is obtained when compared to the rates obtained with non sex-sorted sperm (Seidel, 2014). The P/AI of females inseminated with sex-sorted sperm may be altered by their shortened lifespan in the uterus, decrease in number of sorted sperm per straw and bull fertility (Sales et al., 2011). The diminished lifespan of the sex-sorted sperm in the female reproductive tract, as a result of mitochondria modification and DNA fragmentation, could change the optimum interval to perform AI relative to ovulation (Rath et al., 2013). Thus, similarly to what is observed when conventional semen is used, P/AI of females inseminated with sex-sorted semen is dependent on the fertility of the bulls, animal categories (lactating cows or cyclic heifers), and management across different farms. Consequently, the major commercial recommendation for the use of sex sorted sperm still has been in heifers after detection of estrus, especially due to their higher fertility (Burnell, 2019).

Under Indian condition it is required to standardize the lower dosage of spermatozoa, site of deposition for AI with good conception rates in our conventional system. There is also an extensive demand to develop instruments to transfer sex sorted spermatozoa non-surgically and develop skill in the above area to carry out good results. The main target should be focused to use of sex sorted spermatozoa in good quality heifers and the healthy cycling females in good body condition with excellent reproductive and productive performance to achieve good results and diligence with thawing and handling.

Advantage of using sexed semen in dairy cattle –

 

The principal benefit of sexed semen is to produce a calf of a specific sex. Normally, averaged over thousands of animals, 49% of calves born will be heifers, and a few of these will be sterile freemartins (Seidel Jr, 1999). Supply of replacement heifers in dairy farms is a major issue in commercial ventures. Among successful pregnancies resulting in number of calves born, approximately 90% heifer calf can be produced by using sexed semen. This may be utilized to expand herd size and production. With these increased availability of replacement heifers which thereby reduces dairy heifer purchase and sale prices. As milk production is a sex limited trait and males are not usually maintained by farmers for breeding purpose use of sexed semen will increase with inherd growth and production. Alternatively these heifers may be sold as calves, which would increase incomes compared with the sale of lower value dairy bull calves. This also enables rapid herd expansion without the risk of introducing diseases that occur with purchased animals (Seidel Jr, 1999). By using sexed semen, selection intensity can be increased by choosing genetically superior dams of replacements which accelerate the rate of genetic gain in dairy herds (Weigel, 2004 and Khalajzadeh et al., 2012). It is possible to reduce the incidence of difficulty in first calvers (heifer calves are lighter than male calves) and additional replacement heifers for herd expansion may offer benefits in terms of improved biosecurity by increasing herd size while maintaining a closed herd (Weigel, 2004).

Demerits of sexed semen technology

• High cost of maintenance of sexed sorting machine like flow cyotmetry and lower sorting speed and efficiency
• Higher cost  of sexed semen then conventional semen
• Delayed sexual maturity in heifers under Indian condition
• The conception rate with sex sorted semen is 10–15% less than conventional semen, which is more detrimental condition in our country where total coverage of AI is less than 25% of breedable population (Abdalla et al., 2014).
• Sexed semen contains only 2–4 million sperms/dose as compared to conventional semen which contains 20 million sperms/dose which will be challenge under Indian field condition
• Sperm from some bulls had higher tolerances for sorting, freezing and thawing than from other bulls also every bull’s semen cannot be sexed due to inherent abnormalities in the sperm cells.
• Lack of skilled manpower.
• Lack of awareness about sex semen to farmers. The major problem is that it needs highly specialized, non-portable equipment which is quite costly for routine use.

 

Constraints of sexed semen technology

1. High cost: The machines costs around 4 to 5 crore and along with it royalty should be paid for each dose of semen produced.

2. Commercial availability of the sorting technology: The technology is not fully commercially available. The firm holding patent of orienting nozzle does not sell it and it is a major hindrance.

3. Lower sorting speed and efficiency: The sorting speed of the machine is 6000 sperms per second (Johnson, 2000) and if we run it for 24 hours maximum doses (2 X 106 sperms per dose) which can be produced is 259.2. If frozen semen is produced then it will be 129 doses (4 X 106 sperms per dose). From an ejaculate 30% sperms will be rejected during the sexing process due to non- detecting precisely for difference in DNA content and out of the detected sperm 50% will be Y bearing; so semen doses harvesting from a bull of good genetic merit will be reduced by 70% through this technology.

4. Low conception rate: Conception rate is 10-20% lower in sorted sexed-semen compared to conventional semen (Norman et al., 2010). The lower conception rate in sexed semen may be affected by several factors but the primary factors are low dose rate and physical or chemical stress on sperm during sorting process (Frijters et al., 2009). The sorting stresses include high dilution rate, staining with the dye, mechanical forces, UV laser light beam, and higher fluidic pressure during projection into the collection tube and centrifugation (Garner, 2006). In addition, site of deposition of semen in uterus also affects the CR in sexed semen. Conception rate is more when sexed semen deposited in body of uterus (45%) rather than horn of uterus (32%) in buffaloes heifers (Campanile et al., 2011).

5. Need for standardization in Indian conditions: The technology of sexed semen being patented and used in the western countries, the standardization and production caters mainly to the Bos taurus dairy and beef breeds. Although the technology is claimed to have been tried and used in Bos indicus breeds like Gir in Brazil and some states have adopted the use of sex semen, its use under Indian tropical condition is still to be verified as impact of adopting the technology for genetic improvement of cattle has not been reported. There is a need to standardize the lower dosage of spermatozoa and site of deposition for AI with good conception rate under Indian conditions. Moreover, attribute that differs among the sperm of mammals is the shape of the head. The effectiveness of utilizing DNA content differences between the X- and Y-chromosome carrying sperm depends not only on relative DNA differences, but also on the ability to precisely orient these gametes at the time of measurement in the flow cytometer/cell sorter (Garner, 2006). This makes sex-sorting of sperm not only different for each mammalian species and breed. Hence, standardization of the technique with respect to different breeds of indigenous cattle and buffaloes is also required.

6. Low number of elite bulls: The low number of elite bulls will limit the options for semen sexing in genetically superior bulls.

7. Lack of skilled manpower: Experienced and proven AI technicians are required for inseminations. Furthermore, developing such a scheme for tropical developing countries is constrained by small flock size, indiscriminate mating and absence of pedigree and performance recording.

8. Lack of good quality ejaculates from indigenous cattle and buffalo Practices for optimization the conception rate on using sexed semen

i. Use sexed semen only in herds where the AI pregnancy rates with conventional semen are consistently 60% or more.

ii. Use only in healthy cycling females (heifer and cows). While using fixed time AI, make sure that a high percentage of animals were in heat before doing AI.

iii. Use only experienced AI technicians Be extremely careful with storing, handling and thawing of the straws. Above all, optimal use of sexing technology requires excellent and careful animal management (nutrition, disease control, oestrus detection, semen handling, and insemination technique)

 

CONCLUSIONS

In Indian condition there is need to standardize the lower dosage of spermatozoa, site of deposition for AI with good conception rate in our conventional system. A large number of research need to be carried out to develop this technique in collaboration with other laboratories to make it feasible in India. Furthermore, high cost and fertility related constraints limit its wider use all over the country. So, the main target should be focused to use of sex sorted spermatozoa in good quality heifers and the cows with excellent reproductive and productive performance to achieve good results. There is also immense requirement to develop instruments to transfer sex sorted spermatozoa nonsurgically and to train the skilled manpower in above area to achieve good results. As this technology has much more advantages, so, it is being enthusiastically accepted in many regions of India and it is expected to bloom further in other regions too with decline in cost.

Indian Dairy farmers have started using the sexed semen technology to deal with upcoming challenges in the future Dairy Industry.However, reduction in sperm concentration in sexed semen straw to 2 million viable motile sperm leads to pregnancy depression by 15-20% as compared to conventional system, but fetal female sex ratio is close to 90% with sexed sperm. Optimum fertility from low dose sexed sperm may only be achieved with bulls of high fertility and good managemental practices. Indian farmer needs to be educated and supported financially to adopt sexed semen nationwide. Dairymen could produce more female calves and by this lessen the chance of dystocia at calving, reduce biosecurity risks and genetic improvement at a faster rate. Sexed sperm will cost more and will require greater cattle management and AI breeding skills. More research is needed for sperm sorting efficiency and on large-scale field trials to improve pregnancy rates of low dose, sexed sperm. The Indian government announced, dairy farmers will be provided with sexed semen for Rs 100 per vial by 2020 so that buffaloes and cows produce female calves only.

DR. B. MATHUR, DAIRY CONSULTANT.