Cryopreservation of living cells dates back to 1776 when Lazzaro Spallanzani froze stallion sperm cells. During ensuing years, live offsprings from previously frozen embryos were produced in cows, buffaloes, rabbits, sheep, rats, hores, antelopes and baboons. Use of superior gamets is critical for animal improvement and development programs. Livestock gametes technologies include production, preservation and utilization of gamets viz. semen, oocytes and embryos from individuals with desired traits for faster multiplication and conservation of rare germplasm.

 Video on cryopreservation of Buffalo Bull Semen

•  Semen Freezing: use of frozen semen for A.I. is well established technique for livestock improvement. Buffalo semen can be frozen well if motile spermatozoa are more than fifty percent. The conditions for transport and maintenance of frozen semen in field need to be improved. Well-equipped field staff can be definitely made an impact of this technique in improving the buffalo productivity.
• Oocytes Freezing : Continuous availability of viable developmentally competent oocytes is critical for progress of in vitro embryo production and related biotechniques. Hence, feezing of unfertilized oocytes generate a readily available source for their use on requirement. Since oocytes are single cell therefore, any damage may prevent their development into healthy embryos. The freezing of oocytes is more tricky and difficult than embryo or semen.
• Embryo freezing: The technique of embryo freezing is better established than oocytes freezing. However, it also needs refinement for getting better pregnancy rates following thawing and transfer to recipients as per requirement, thus avoiding the need for estrus synchronization. Embryo freezing makes it easy to transport quality germplasm throughout world. Embryo transfer is a technique of choice for cattle breeding in some countries but in buffaloes this also needs dedication and high quality infrastructure to take advantage of this technique.

Selection of sex of offspring in livestock depends on the economic importance and utilization of species. In India male bull calves are preferred for use in agricultural operations. With increased mechanization of agricultural operations, there is minimum requirement of these males. In dairy industry, female calves are preffered over males and with the development of techniques like AI large number of females cab be inseminated with semen from limited number of bulls. Sex selection techniques are now quite useful in selecting sex of young ones in economically important livestock species. Sexing of embryos can be done with PCR technique by taking biopsy of few cells from embryos and using a pair of cattle specific BRY1 primers for amplification of male specific fragment on buffalo Y Chromosome. The assay can be performed in less than 4h and embryonic sex can be determined accurately using only 1-2 blastomeres.

Calves of desired sex can be produced by inseminating animals with pre-sexed semen. Separation of male and female spermatozoa can be done by cell sorter using weight difference in male and female i.e. X and Y chromosome bearing spermatozoa. Fetal sex determination can also be done with noninvasive technique of ultrasonography around day 55 of gestation in buffaloes.

Introduction

Artificial insemination (AI) is a technique in which sperm are collected from the male, processed, stored and artificially introduced into the female reproductive tract at proper time for the purpose of conception. AI has become one of the most important techniques ever devised for the genetic improvement of farm animals. It has been most widely used for breeding dairy cattle and buffalo.

History

The history of the development and use of artificial insemination in domestic animals is a long and varied one.

1300 A.D- The first reported use of AI, although not documented. In Arab countries artificial insemination was carried out in Arab horse breeders. According to an Arabian book published in 14th century; an Arab chief of Darfur put a ball of cotton in the vagina of a mare which was recently bred by a famous stallion belonging to an enemy cheiftan during night time. After 24 hours, he then hurried to home and introduced the cotton ball into vagina his own mare. The mare became pregnant and gave birth of a foal.

1677 – A major technological breakthrough in the study of reproductive physiology was made by a Dutch Scientist named van Leeuwenhoek, who developed a simple microscope. A medical student suggested to van Leeuwenhoek that semen might contain living cells using his microscope; van Leeuwenhoek observed semen and discovered that it contained small particles that moved about. He referred to these particles as “animalcules” and published a paper on his observations in 1677.

1780- An Italian physiologist, Lazzaro Spallanzani reported first successful use of AI. After success with several amphibian animals, he started experiment with dog. Dogs were confined in his house. When one bitch manifested the signs of heat, he used semen at body temperature to inseminate the bitch. Sixty-two days later she gave birth to three pups. He is also called “Father of modern artificial insemination”.

1803- Spallanzani reported cooling of semen prolong the sperm life. Further development did not occur for a very long time.

1914- G. Amantia, professor of human physiology at University of Rome developed the first artificial vagina for collection of semen from dog.

1937- Danish veterinarians developed the first rectovaginal/cervical fixation method of AI.

1940– Philips and Lardy developed egg yolk phosphate diluter for preserving fertility and motility of refrigerated bull spermatozoa.

1941- Salisbury et al. developed egg yolk citrate diluter.

1948– Sorenson first time used large sized straws (12mm) made up of polyvinyl chloride.

1949- Polge, Smith and Parkes discovered cryoprotective effect of glycerol in frozen semen technology. This is most important milestone in the history of artificial insemination.

1951- Steward reported the birth of first calf from insemination with frozen semen in cooperation with Polge and Smith.

1963- Nagase and Niwa developed the technique of freezing bull semen in pellet form in Japan.

1964- Cassou improved the straws by reducing their size and named it as medium French straws. The size of the straw was 135 mm long and 2.8 mm diameter with 0.5 ml semen capacity.

1968-Cassou further reduced the size of the straws to the diameter of 2 mm with a capacity of 0.25 ml and named it as mini French straws.

1974- Japanese Scientist Nishekawa et al. first time frozen the semen in liquid helium at -265^oC.

1972-A plastic straw called mini tube or German straws or ‘Lanshut system’ was developed in Germany. These straws are sealed by metallic or glass or plastic balls.

1960-Adler developed the first technique for freezing of semen in straws using liquid nitrogen vapour.

Work done in India

1939-In India, first time, AI was done by Sampat Kumaran at ‘Palace Dairy Farm Mysore”. He inseminated large number of Halliker cows with semen of Holstein Friesian and got 33 cows pregnant.

1942-A pilot project was started at Indian Veterinary Research Institute (IVRI) to study the feasibility of AI under the guidance of Dr. P. Bhattacharya with the team consisting of Dr. S.S. Prabhu, Dr. D.P. Mukherjee, Dr. S.N. Luktuke, Dr. A roy and Dr. Garjan Singh. This team approved that this technique could be used in Indian Condition since then, this technique has come into general use as a regular practice of breeding for cattle and buffaloes.

1942-Four regional centres were established at Bangalore, Calcutta, Patna and Montgomery (Now in Pakistan) by Govt. of India.

1943-The first buffalo calf through AI was born at the Allahabad Agricultural Institute.

1951-56-In the first five-year plan (1951-56) the Government of India introduced 150 key village centres to improve cattle and buffaloes in this country.

1956-61-The second five-year plan (1956-61) gave a boost to AI work by implanting it in 400 key village centres.

contributed by Pradeep Kumar, Dharmendra Kumar,Indrajeet singh and Sunesh

• Semen
• Site
• Time
• Technique

• Follical Dynamics
• Pregnancy
• Infertility
• Treatment
• Fetal Sex And Age

In embryo transfer both male and female gametes can be selected from the superior animals and resulting embryo shall have genetic gain from both the parents. There are two major source of getting embryos from live animals Single Ovulation- Embryo Transfer (SOET) and Multi- Ovulation Embryo Transfer (MOET).

IVEP  SOET  MOET

Cloning mainly comprise of reproductive cloning and somatic cloning depending on the resource cells used.

• Reproductive Cloning : Use of blastomeres of an embryo to produce more embryos is known as reproductive cloning. This uses the totipotent power of embryonic cells at early stage of development. This technique can be used for multiplication of superior animals at faster rate.
• Somatic Cloning : The cloning of animals received much attention in 1997 and 1998, with the production of lambs and calves by nuclear transplantation of differentiated or immortalized fetal and adult cells into enucleated metaphase || oocytes. Although the technique is not efficient, the ability to clone animals from differentiated cells has changed our understanding of the irrevocable nature of cell differentiation and germline totipotency.

Nuclear Transfer (NT) is one of the more recent biotechnological procedures for cloning, where the nucleous of an oocytes or zygote is replaced with the nucleous of developments have stimulated interest in this technology globally, starting with the highly publicized birth of ‘Dolly’ cloned from the nucleus of a somatic cell. Subsequent successes were reported in severable species of echonomic interest. NT could be useful in multiplying high value transgenic animals, extreme high producers for traits such as milk in dairy animals, specific disease resistance or environmentally adaptable animals and for conservation of wild and domestic species at the verge of extinction. Yet, the success rates of cloning by nuclear transfer remain very low even in the most researched species and require further scientific input to improve the technique.

Success of NT depends upon different steps starting from proper enucleation and reconstruction procedures, obtaining suitably synchronized stage of development of donar embryonic cell/embryonic stem cells/somatic cells followed by fusion of donar nucleus with the recipient cytoplasm. This has to be done in such a way that nucleus introduced into the enucleated, timed and prepared metaphase|| oocytes is capable of re-expressing its entire genome. Susequently, optimum culture conditions are required to support development of the nuclear- transferred zygote to a syage suitable for transfer to recipient animal for completion of gestation.

Failures are not uncommon, resulting from abnormal chromatin of donar cells, inappropriate or incomplete nuclear or cytoplasmic DNA and protein reprogramming, damage during micromanipulation procedures for enucleation and donar nuclear transfer, apart from culture process inadequacies. Hence, there is need to understand intricacies of the cytoplasm- nuclear interactions and to refine the procedures for obtaining donar nuclei at predetermined stage in the cell cycle for optimum success. Research is needed to identify the cell types and conditions most likely to provide totipotent nuclei for transfer.

First transgenic livestock was produced in 1985 using microinjection of foreign DNA into pronucleus of Zygote. This technique is having many inherent shortcomings like low efficiency and random integration into host genome. Various alternative new methods are sperm medicated DNA transfer, ICSI of sperm heads carrying foreign DNA, inserting viral vector in oocytes or embryo and use of nuclear transfer. Further development include use of large genomic construction to optimize expression, application of RNA interference to knock down specific genes.

Numerous proteins have been produced in large amounts in the mammary gland of transgenic sheep, goat, cattle, pig and rabbit. Trials on antithrombin|||(AT|||) productionin mammary gland of transgenic goat have been completed and the recombinant product has been approved as a drug by European Medicines Agency in August 2006. Successful drug registration of AT||| demonstrates the usefulness and solidity of this approach and will accelerate the registration of further products from this process. Other recombinant proteins from the udder are alfa-antitrypsin and tissue plasminogen activator. To ensure safety of recombinant products guidelines developed by Food and Drug Administration of USA require monitoring animals’ health, valditation of the gene construct characterization of isolated protein, as well performance of transgenic animals over years.

 The biotechnologies applied in buffalo reproduction are restricted so far to AI, with very few attempts in ET, IVF and sexing technologies. Actually application of such technologies to buffalo reproduction requires addiotional basic research to understand low success rate. Superovulation and embryo transfer, though less successful, but can be applied at least in bull production. In vitro embryo production rate has improved significantly and in vitro fertilization can be used to test the fertility of bulls. Freezing of embryos is successgful and oocytes freezing is in progress and comparable with other species. There is scope for working on stem cell, somatic cell and multiple generation cloning. When the procedures become more efficient the possibility exists for incorporation of embryonic stem cells as donor nuclei. Developmental potentional of embryonic stem cells needs to be studied. These biotechnologies in the buffalo offer an enormous opportunity for genetic gain, as well as increased efficiency of food production for both vegetarian and non- vegetarian population.