Sexed semen: challenges and opportunities for Indian dairy industry

Introduction

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

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.

Importance of sperm sexing

1. Unwanted male calves can be minimized which help the farmers to save resources wastage on management of male animals.
2. Helpful in genetic improvement in case of selection of best female for different traits.
3. 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.
4. For progeny testing program, it ensures required number of daughters under short time, thus it increase genetic gain.
5. During the sorting process, dead sperms are removed, which shows the existence of only viable sperms.
6. Reduce the chance of dystocia cases by avoiding production of male calves by use of sexed semen
7. 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.

Demerits of sexed semen technology

1. High cost of maintenance of sexed sorting machine like flow cyotmetry and lower sorting speed and efficiency
2. Higher cost  of sexed semen then conventional semen
3. Delayed sexual maturity in heifers under Indian condition
4. 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).
5. 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
6. 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.
7. Lack of skilled manpower.
8. 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.

CONCLUSIONS

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.

Conflict of interest

The authors declare that they have no conflict of interest.