Prospects of sexed semen of cattle and buffalo in India

                                           Dr R K Patel

Introduction

Artificial Insemination (AI) has been accepted as most important technology worldwide for the animal improvement programmes and being widely used for breeding dairy cattle and buffalo in India. AI is a popular, simple and inexpensive treatment of infertility in farm animals, in which the sperms from male is collected and introduced artificially into the reproductive tract of the female for conception. The history of the development and use of artificial insemination in domestic animals is very old and varied. An Italian physiologist reported first successful use of AI in dog during 1780.  In India, the first AI was done in Halliker cows with semen of Holstein Friesian and got 33 cows pregnant in animal farm at Mysore during 1939. During 1950s, key village schemes were introduced by Govt of India to improve animals through AI. The biggest advantages of AI are to utilize quality sires effectively for a large population of females and prevent the transfer of venerable diseases which can otherwise transfer in natural course.

Present status of AI

With a breedable population of 199.1 million cattle and 105.3 million buffalo (source: Annual report 2011-12, Ministry of Agriculture, Govt of India), a scientifically planned programme to improve milk production to meet the demand for milk by the rapidly growing population in India, is required. At present, there are more than 50 semen stations in the country producing approximately 70 million doses which are not enough to breed more that 25% breedable population of the country. To cover the whole dairy animal population, we need to increase and strengthen present sperm stations, produce sufficient breeding bulls for AI and replace old bulls regularly. Theoretically, the frozen semen produces 50% male and 50% female progenies. However, to increase milk production to fulfil the per capita requirement, we need more female births that can only be achieved by the sexed semen.

Conventional methods used for sexing of semen

In ancient time, people used to suggest that right testis produces male whereas left produces females. In normal condition, the male progeny producing sperm bears Y chromosome whereas and female producing sperm bears X chromosome. The quest to identify or separate male and female sperms remained great interest among research communities for many years and therefore various methods were adopted in past. Scientists worldwide tried to sex the sperms by size, weight and density (Bhattacharya, 1962); swimming capability/speed (Erricsson et al., 1973) electrical surface charges (Shirai et al., 1974) surface macromolecular proteins, differential effects of pH etc. Scientists also tried to separate male and female sperms by atmospheric pressure; such as sedimentation, electrophoresis, centrifugation, sephadex filtration etc. However, none of these methods was able to significantly separate the viable sperms capable to fertilize. Hence, none of these methods became popular to achieve the goal of separation of X and Y bearing sperms.

Current status of sexed semen

It is now possible to predetermine the sex of offspring in number of species with the accuracy of 85-95% (Shidel et al., 1999; Johnson, 2000). Thousands of offspring have been produced now by using AI with sexed semen (Seidel and Garner, 2002). Many companies, institutions, American cooperatives and breeders are producing, practicing and commercializing the sex semen worldwide. However, in India, Puschim Banga Go-Sampad Bikash Sanstha (PBGSBS), Haringhata (WB) initiated sorting of semen by semen sorter installed during 2009. It is now producing 40-50 numbers of frozen semen straws of sexed semen per day. They claimed to produce the first predetermined sexed male calf named Shreya born on 1st January 2011. They also claimed to produce three female calves at bull mother farm of PBGSBS. Some of America’s largest artificial insemination cooperatives are peddling to dairy farmers in Punjab providing a breeding solution that promises 90 per cent production of cows. The state government has imported 5,000 sexed semen straws to produce female calves (source: Mail Today, Chandigarh, May 16, 2012). Last year, Worldwide Sires partnered with the Punjab Animal Husbandry Department, the Punjab Dairy Development Board and the Ludhiana-based Dairy Farmers Association has decided to supply 88,000 doses of sexed semen to Punjab farmers. This year, the cooperatives are reaching out directly to dairy farmers in the state for the requirement of sexed semen doses.

Sperm sexing by Flow Cytometry

The most advanced, very effective and producing viable sexed sperms, is flow cytometry. The basic principle of the flow cytometry is based on the differences in the DNA contents of X and Y bearing spermatozoa as Y chromosome in most species is smaller than X chromosome. Therefore, the content of DNA of X chromosome is more than Y chromosome. The difference in the DNA contents differ among species (Moruzzi, 1979) and it varies from 3.6-4.2% (Johnson, 1992; Johnson and Welch, 1999). Ram, Rabbit, Bull and Boar spermatozoa have a difference of 4.2%, 3.9%, 3.8% and 3.7% respectively. In Bos indicus, the average X-Y sperm difference is 3.73%. Whereas, differences in DNA content for Murrah and Nili-Ravi buffalo were 3.59% and 3,55% respectively (Lu et al., 2006; Mir and Kumar, 2012).

For sorting of the spermatozoa by flow cytometry, two kinds of dyes or stains are used; i) Hoechst 33342 which is fluorescent dye binds with DNA of live spermatozoa (Johnson et al., 1987) and ii) non toxic food dye that penetrates into the non-viable spermatozoa due to damaged membrane and reduces the binding intensity of the fluorescent dye in the dead. The food dye thus helps to eliminate the non-viable spermatozoa from the sorted population and increases the viable count in the gender selected spermatozoa. The stained spermatozoa after incubation at 340C are passed through a miniature nozzle in thin stream under pressure (40-50 psi) which is equipped with laser beam (blue light). The laser beam fluoresce the stain bound to the DNA of the spermatozoa. X chromosome- bearing sperm fluoresce with 4% more intensity than Y- chromosome bearing sperm and that is the principle of separation of sperms. Droplets carrying viable spermatozoa are charged (+/-) depending on the amount of DNA before passing between the appositively charged plates. The droplets contain more than one sperm. Dead sperm stained by food dye, or those where DNA content could not be measured accurately or not charged are not sorted and goes as waste as shown in figure. Samples are then collected in three containers, X and Y- chromosome bearing and unsorted. This process allows sexing and collection of about 40% of the sperm going through the sorter at a speed of approximately 90-100 km/h when they exit the nozzle. Discrete fluorescence signals from two detectors at 90Angles to each other are produced, and the information is processed by computer. Thus, at an event rate of 20,000-25,000 total sperms/second, nearly 4,000-5000 live sperms of each sex can be sorted simultaneously. The current system can produce approximately 10-13×106 live sperm/hr of each sex with 85-95 % accuracy (Seidal et al., 1999).

Strengths and risk factors of flow cytometry

Strength: i) Thousands of offspring have already been produced worldwide via Artificial Insemination with spermatozoa sexed by flow cytometry and cell sorting. ii) Accuracy of sexing can be obtained up to 95% in most species iii) Sorted semen can produce progeny of desired sex; females for milk production, males for beef production and genetically superior males for improved breeding and iv) Demand of sorted semen including India is very high as no organization is supplying the same on commercial basis.

Risk factors: i) Approximately 40-50% losses of spermatozoa during sorting processes; residual loss of spermatozoa in staining tube, spermatozoa discarded due to malorientation, discarded dead spermatozoa, losses of spermatozoa in the supernatant-15% after centrifugation, Loss of spermatozoa during filling and sealing and ii) poor insemination practices using sorted frozen semen may question the technology.

Conclusion

As we know animal breeders have already exploited genetic potential of milch animals with the help of artificial insemination, upgrading, selection of pedigreed animals, improved breeding practices, crossbreeding and marker assisted selection (molecular techniques) etc., no options are now left with the scientists/breeders to increase milk production to meet the demand of ever growing population worldwide. Increase the population of potential milch animals (females) to produce milk many folds is the only option left with us. Therefore, using sexed semen in our country would be   a boon for milk production.