Preface There is a great paucity of texbooks in the field of veterinary science written by Indian authors as reference and textbooks. An attempt has been made by the editors to produce a text on Artificial Insemination and Treatment of Infertility in Dairy Animals. It is a handy work of 17 well experienced faculties drawn from different departments of higher learning. Most of them are actively engaged in under-gradate and post-graduate teaching with considerable expertise. Adequate care has been exercised by the editors to incorporate all the aspects of artificial insemination and infertility so that it can serve as a real guide to the students and veterinarians in minimizing the possible economic loss to the dairy animal owners and dairy industry.

Estrous cycle After puberty the female enters a period of reproductive cyclicity, which continues throughout most of her productive life.  Estrous cycle is the rhythmic sexual behavioral pattern exhibited by the female, which begins at one estrus (heat) and ends at subsequent estrus.   It is the period between two estruses of the regular cycle.  The reproductive activity of the female is maintained by the co- ordinated functioning of different organs of reproduction (Table: 1.1). Estrous cycle provides repeated opportunities for the females to become pregnant. When conception does occur, the female enters a period of anestrous during pregnancy, which ends after parturition (giving birth) and uterine involution (repair and returning to normal size).

In our country livestock are mostly dependent upon cereal by-products or the crop residues such as straws, stovers etc., which are low in nutritive value. According to the National Commission on Agriculture (1989-90), there is a shortage of 40% of concentrates, 20% of dry fodder and 40% of green fodder for meeting the nutritional requirements of livestock. Consequently the livestock is surviving on low weight, low energy and thereby leading to problems in the production and reproduction. Nutritional deficiencies as well as imbalances are implicated as the cause of infertility in animals. The clinical signs observed with the deficiency of a specific nutrient as variable depending on the degree of the deficiency. The information on the important relationship between nutrition and health is necessary for recognizing and correlating the reproductive problems.

Livestock diet or ration may be made up of one or several ingredients depending on the species of animal, their physiological state, source of feed and its nutritional composition, nutritional requirements of the animals and voluntary intake of the feed. Chemical and nutrient composition with each ingredient varies tremendously and no single feed ingredient is complete in its nutrient composition with respect to animal’s requirement.  Therefore, animal feed has to be balanced properly to supply required nutrients in appropriate quantity.  Feed formulation is an art of quantitative application of science of nutrition for the optimum supply of all the essential nutrients through actual feeding of animals. This exercise may be as simple as mixing of few ingredients with simple mathematical calculation or preparation of least cost balanced ration by computer programme.  Since the expenditure on feeding is substantial in livestock production, feed formulation needs special attention of nutritionists or feed technologists. The instruments and equipments used for feed formulation and estimation of micro minerals and ingredients like Feed grinder and mixer of capacity 75 kg, Fibertech for fiber estimation, Hammer mill-particle size reduction, Mixer and grinder for feed and fodder (complete feed preparation) of capacity 250 kg, Roughage processing-Chaff cutter, Soxhtech for fat estimation, UV-vis-Spectrophotometer and Voltameter for micro mineral estimation are shown in Fig. 3.1 to 3.8.

India has a mega animal bio-diversity, which is manifested in the form of availability of a large number of species, and a variety of breeds even within species. India is bestowed with rich and varied domestic animal biodiversity having a total of 32 breeds of cattle and 10 buffalo breeds. The diversity of livestock is not only accidental, nor purely natural; it is more the outcome of thousands of years of continuous selection. The trends in livestock population over the last three decades have shown that, there is significant increase in the population of buffaloes i.e. from 60.5 crore in 1973 to 96.9 crore in 2003, while cattle population has increased from 180.1 crore to 226.1 crore in the same period. The share of buffaloes, in the bovine population increased from 22 to 32 per cent while that of cattle has shown declining trend. Within the cattle, there has been marked shift from work animals towards milch animals which has resulted in loss of important draught breeds of cattle. Further indiscriminate crossbreeding has resulted in significant decline in number of important milch breeds like Sahiwal.

Faulty estrus detection is the most important problem in reproductive management.  There are two requirements for good estrus detection: a skilled observer must spend sufficient time to make observations and cows must show overt signs of estrus during the period of observation.  There are various ways to detect the estrus signs in cows and buffaloes as detailed below. 1.    Palpation of reproductive organs:  Routine rectal examination of all cows between 30-40 days after calving and individual cow that has not been inseminated within two months after calving should be encouraged.   This is to confirm that the reproductive tract is normal and to predict when the next estrus will occur or to identify cows for prostaglandin treatment when estrous cycles are occurring, but estrus has not been detected.  2.     Teaser bulls:  Teaser bulls are useful in identifying estrus in large number of animals especially buffalo cows.   Teaser bulls can be prepared by vasectomy or by applying apron to a bull.  3.     Heat detector animals:  Sexually active animals like bulls, cystic cows, hormone treated steer, cows and heifers can be used to identify estrus cows.    They may be fitted with halters containing ink- filled reservoirs and ball paint pen type devices (chin ball markers) that will mark animals that mounted or they can be used without these devices to increase the sexually activity and make visual detection programs more effective.

After the Second World War, the 1940’s and 1950’s was the wide spread establishment of artificial insemination and the introduction of frozen semen in cattle breeding.  In the late 1960s came revolution in measurement of hormones in the body fluid with the introduction of sensitive radio-immuno-assay (RIA) techniques, which provided a new approach to early pregnancy diagnosis, emerging around the same time were early forms of the ultrasonic which could also employed in the detection of pregnancy. The 1970’s were to see embryo transfer and freezing of embryos in cattle becoming commercial realities and their incorporation into increasingly effective breeding improvement programme with the availability of prostaglandins (PGs) and Gonadotrophin releasing hormones (GnRH) that decade also witnessed new possibilities for estrum control in cattle and horses.  Few years later came the ability to produce the cattle embryos in the laboratories in large numbers and at low cost.

Synchronization of estrus is making a group of cows to come into estrus at the same time.  Effective estrus detection requires much time, labour, skill and expense and it is a major limiting factor in artificial insemination programme in cattle.  It was the principal stimulus that led to research in the development of progestational and prostaglandin compounds.  Advantages 1.     Improve the efficiency of heat detection 2.     Management tool, better planning of breeding activities and wider use of artificial insemination  3.     Solve problem of unobserved estrus 4.     Maintain short calving interval 5.     Overall improvement in herd productivity and reproductive efficiency 6.     Controlled lactation 7.     Management of replacement heifers 8.     Useful in embryo transfer programmes 9.     Reduce labour 10.     Possibly reduce involuntary culling for reproductive reasons

Buffaloes, though have great production potential, do suffer with many reproductive problems viz.  delayed age of puberty and  sexual maturity, weak / silent estrus, longer inter-calving period,  seasonality in breeding, poor libido  and poor semen quality. Little information is available about the buffaloes as compared to cattle because all the time we try to apply the knowledge available about the cattle to the buffalo.  Some comparative reproductive parameters in cow & she buffalo & cow bull and buffalo bull are depicted in Table 8. 1 and Table 8.2 respectively.

The ultrasonography is a technique which uses sound waves of very high frequency to scan the various organs of the body. The ultrasound waves of 2-10 mHz frequency are used in veterinary practice. A quartz crystal when stimulated by short pulse current ultrasound waves are emitted. The ultrasound waves emitted are directed towards a specific target organ are reflected back as echoes. The echoes are received by the same quartz crystal and are processed in the electronic circuit and finally displayed as image on the monitor.

Laparoscopy is an operative procedure performed through a key-hole opening with a rigid endoscope that allows visual inspection of organs.  The laparoscopy helps in arriving diagnosis of various reproductive and digestive disorders quickly.  By laparoscopy clinician can visualize the external surface of the abdominal organs.  Tumours, abscess, cyst, lesions, growth, hemorrhage, adhesions of the organs in the abdominal cavity can be readily diagnosed by this technique.  Aspiration of follicular fluid, biopsy of lesions and sterilization are commonly employed maneuvers by this technique.  It is having both diagnostic and therapeutic value.  The procedure consists of placement of transabdominal rigid endoscope after establishment of pneumoperitoneum in the body cavity.   The images of different organs are displayed on the TV monitor when the laparoscope is moved within the peritoneal cavity with the illumination through halogen light source.

Non-specific uterine infections reduce the reproductive efficiency of livestock.  They are called nonspecific because the initial colonizing bacterium is not known and the specific bacteria causing the signs of infection are not known.  Even though numerous bacteria, in a variety of combinations, have been isolated from infected uteri, Corynebacterium pyogenes and Escherichia coli are usually associated with uterine infections in cattle and sheep.  Empirical therapeutic approach, prescriptions without scientific validation (extra-label use) or inadequate clinical judgment itself to ascertain the nature of uterine infection, obviously due to lack of diagnostic tools are the prime reasons for therapeutic failure for the unsuccessful treatment of uterine infection by large animal practitioners.

Buffalo plays an important role in Indian livestock economics.  India has more than the half of the world buffalo population, contributing 50% of total milk production of the country.  The buffalo is said more productive than an average cow due to the higher fat percentage (7%) in the milk, better feed conversion ability and disease resistance.  Buffaloes have lower reproductive efficiency in the form of delayed age of maturity, longer calving interval, weak / silent estrus, seasonality of breeding, poor libido and poor semen quality. A large number of buffaloes do not conceive due to some gynaecological problems and are being slaughtered every year.  The most commonly observed reproductive disorders are detailed below.

Infertility means reduced fertility or the cow is ultimately capable of becoming pregnant and giving birth to a live calf but the interval may be much longer than 12 months.  I. Problems leading to infertility 1. No signs of estrus When the animal is pregnant and for a short time after calving the cows may not show estrus signs. A thorough palpation of the ovaries and tubular genital tract should be performed and a repeat examination may be necessary.

A typical repeat breeder cow or buffalo is an apparently normal cycling cow / buffalo with normal genitalia which has failed to settle to services or inseminations during three or more consecutive estrus periods. These animals can neither be considered as sterile nor can settle to subsequent services.  Age of the repeat breeder should not have exceeded beyond 12 years. The causative factors for repeat breeder animals fall in two main categories as failure of fertilization and early embryonic death.

Production is based on reproduction and health of reproductive system. Economy of livestock industry is directly attached to the maintenance of high fertility in the herd so controlling the reproductive diseases in cattle is a must.  Reproductive health may be disturbed by reproductive infections like Brucellosis, Leptospirosis, Infectious bovine rhinotracheitis and Bovine viral diarrhea.

There is no rapid direct test to determine fertility of semen sample. During freezing and thawing of semen 20-80% of the spermatozoa become immotile or die. In addition to this, there may be damage to spermatozoa by disintegration of sperm cell membrane and acrosome leading to loss of intracellular enzymes.   The only reliable method to evaluate the semen is to inseminate a cow and get a calf at the end of gestation period.  Now-a-days new methods have been developed for in-vitro assessment of fertility of spermatozoa.  Some of these methods are exhaustive, time consuming, need for a full fledged analysis and difficult under field conditions.

Several cryogenic agents have been used in many countries for freezing and preservation of bovine semen like solid carbon-di-oxide (-79°C), liquid air (-183°C), liquid helium (-268°C) and liquid nitrogen (-196°C).  Liquid nitrogen is 4th coldest substance known and its small amount is converted into a very large amount (near about 700 times) of its volume when exposed to atmosphere.  It is a colourless, odourless, non-irritant, non-toxic, non-caustic, non-corrosive, non-inflammatory and inert gas having boiling point -196°C temperature.  Being a liquid it comes into good contact with the surface of the semen straws and constant storage temperature is maintained throughout the straw and due to these properties LN2 is most widely used refrigerant in the world.

The various steps involved in frozen semen technology are vital as the quality of semen determines the conception rate.  The key factor enabling the successful long term storage of frozen spermatozoa is maintenance at ultra low temperature.  Effective semen storage depends heavily on good identification and stock control.  Frozen semen can be easily damaged in the process of repeated handling to establish identity.

Reproductive process involves many events that have to occur in a well balanced manner if satisfactory fertility rate is to be achieved. Any adverse  effect of  physiological, morphological  or environmental effect on the male  or  female may result  in failure  of  fertility  or reduced fertility  depending  on the severity of the cause. Apart from this, the role of person who stands between the cow and the bull play an important role. The efficiency of the person handling the semen and depositing in the reproductive tract of domestic animal will influence to a great extent on the conception rate in A.I. programme. It is observed that certain bovine population fails to conceive at a single service or series of services.   Some times there is loss of conceptus during gestation or produces a dead calf or weak calf that will die soon after birth.   Consequences of these factors affect cows and may have long calving interval, abortion or sterility, all of which tend to decrease the average daily production of milk.

The choice of hormones and drugs acting on reproductive system are important as therapeutic agents during reproductive disorders like anoestrus, metritis, pyometra, repeat breeder, retention of fetal membranes, delayed puberty, ovarian disorders etc.  The administration of specific chemotherapeutic agent is of paramount importance in treating reproductive disorders in dairy animals.  The dosage, route, frequency and indications of chemotherapeutic agents acting on reproductive system are mentioned in tabular form as below.