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Infertility, not only in farm animals but also in companion animals has remained a burning issue for many decades. Although female infertility is often considered the primary culprit, it is crucial to recognize that male animals are equally responsible for this issue. One can say that the reproductive efficiency of the male animal plays a decisive role to get the maximum productivity from female animals. In the same context, it is very important to study all the aspects related to the bio-physiology of the male reproductive system to understand the causes of male infertility. By comprehending the intricacies of male reproductive biology, andrologists, and clinicians can develop effective remedies to address the issue of infertility in male animals. Moreover, addressing the problem of male infertility in animals along with better use of artificial insemination can have far-reaching benefits, such as improving the production quality of livestock and increasing the availability of companion animals for adoption.

Anatomically the male reproduction system comprises of testes, scrotum, duct system, accessory sex glands, prepuce, and penis. Production of spermatozoa and male sex hormone are the main functions of male reproductive physiology. The Male reproductive system can be categorized as: • Primary sex organ – Testicles • Secondary sex organ – Epididymis, vas deference, urethra, and penis. • Accessory sex glands – Seminal vesicles, prostate, and bulbourethral glands/cowper’s gland. 1. Testes (Paired male gonads) (Singular: Testis or Testicle) • Testes are the primary sex organs. They are situated in the scrotum at the inguinal region with the help of tunics and spermatic cord. Opening of the scrotal pouch into the abdominal cavity is called as inguinal canal. The testis is covered with an extra abdominal tunic named as tunica vaginalis, tunica albuginea, and tunica vasculosa from outside to inward. The fluid filled in between the parietal and visceral layers of tunica vaginalis is responsible for the movement of the testis within the scrotum. The upper end of a testicle is occupied by the caput epididymis and the lower end is connected to the cauda epididymis.

• In male animals, endocrine regulation of reproduction is mainly controlled by pituitary gonadotrophins and gonadal androgens. It is not the same as in females as there is only negative feedback controls in case of male while it is positively as well as negatively feedback control in females. In other words, the synchrony of pituitary gonadotrophins with gonads in male is non cyclic while in female it is cyclic i.e. even after castration (absence of androgen) the pulse frequency of LH and FSH is retained. • Sex Hormones are sex specific e.g. estrogen and progesterone work primarily in females and testosterone in males but sex specificity is limited as far as sexual behaviour is concerned. As estrogen injection in castrated ram recovers the maleness while testosterone injection in ovariectomized female maintain the receptivity for male up to a certain extent.

• To identity the normal fertile, infertile (Temporary loss of fertility), subfertile (reduced fertility), or sterile (no fertility) male animals makes the requirement of breeding soundness evaluation necessary. Infertile and sterile animals prove to be harmful for the economic survival of the dairy industry and should be eliminated from the breeding management system. • The breeding soundness examination is very necessary for the breeding management system as the ultimate goal is the achievement of healthy pregnancies. It is also useful for successful artificial insemination (AI) programs that involve the collection and cryo-processing of quality semen.

Puberty • It is the age at which sexual organs are functionally developed and gonads start responding to GnRH to start steroidogenesis and spermatogenesis. Animal at puberty develops secondary sexual characters with proper libido. • At puberty, the bull is able to produce 50 million spermatozoa with 10% progressive motility in 1st seminal ejaculates. It should be 100 million spermatozoa with 10% progressive motility in 1st seminal ejaculate in case of stallion. • At puberty, gonadotrophic cells are functionally developed in the mature pituitary tends to gonadal maturation. The steroidogenesis starts properly which leads to the development of genital organs and secondary sexual characters.

Libido • It is the willingness & eagerness of male animal to mount and perform complete service. It is sexual desire or drive which is an innate character of male animals. • Libido and mating ability have no direct relation with fertility. Libido is not related directly to sperm production and semen quality. • The libido of an animal depends on androgenic steroid hormone. Vasectomy or cryptorchidism does not affect libido in male animals. • Animals have no or reduced libido that are castrated before puberty. If the animals, that know the copulation, are castrated after puberty, they display the copulation behaviour for longer period. • Reaction time – It is the time between the contact of a male animal with female animal to 1st false mount with erected penis. Reaction time is the good predictor of libido.

• Sexual activity or mating behaviour is innate character of the males. Unlike the female animal, male animal has a nature of sexual receptivity permanently. • Male gonadal steroid androgen controls sexual behaviour. At regular interval of 2-4 hrs, there is secretion of testosterone which converts into estradiol due to aromatization. Due to this very frequent rise in estradiol level, male shows sexual behaviour permanently. In female animals, a rise in estradiol occurs only during estrous. • When in a group of animals, any female can be mated with any male, it is called promiscuous sexual behaviour. Sexual behaviour in some animals is according to their social structure and territorial/home range i.e. male and female share some of their home range for the purpose of mating e.g. Roe deer, Muntjak antelope etc. In some animals, both males and females share whole of their territory for the mating purpose called a harem e.g. wild rabbit. Some animals come into a temporary territory meant only for mating in breeding season e.g. seal.

Fertility is the ability of a male to impregnate the fertile female. The reduced or temporary loss of fertility is called infertility whereas permanent loss of fertility is called sterility. A male with low libido or poor sex drive may have excellent semen quality or a male with good libido may have poor seminal quality. So, libido has no direct relation with the male fertility. Male infertility can be categorized into Impotentia coeundi and Impotentia generandi. 1. Impotentia Coeundi • The condition of reduced or lack of sex drive or Inability to copulate (failure of normal service) is referred to as impotentia coeundi. • It may occur due to immaturity, poor experience to perform coitus, inability or unwillingness to mount, inability to perform intromission or failure of ejaculation. • The following conditions may reflect the immaturity or poor/bad experience of the male animal which leads to impotentia coeundi:

• These are the injuries occurred to male animal at the time of natural mating. These injuries do not occur only to genital organs but also to other organs or muscles. • Most coital injuries occur to male animal with good libido (high testosterone level) but having aggressiveness and poor experience of mating. Such animals tend to quick response in the presence of estrous female. Some of the coital injuries are: 1. Balanitis and Posthitis • Trauma to the penis and prepuce which later on leads to infection and inflammation. • This occurs especially in young inexperienced bulls with great sexual desire • Adhesion may lead to phimosis or paraphimosis condition • IBR-IPV infection from cow genitalia after mating results in pus formation and necrosis of the penis and prepuce. • Prepucial eversion leads to laceration and bleeding.

• These are the bad habits of the male animal due to which ultimately their maximum reproductive potential cannot be utilized. • There are so many vices seen in male animals like stall walking, weaving, cribbing, wind sucking, crib biting, viciousness etc. These behavioural disorders are mostly due to improper managemental practices like abusive handling, poor exercise, irregular semen collection and poor exposure to female etc. These may be also due to hormonal imbalance or nervous disorders. - Some important vices which are more related to reproductive behaviour are: 1. Masturbation (Onanism) • Bulls masturbate by rubbing their prepuce and penis against any object. • Boar masturbate and ejaculate by inserting the penis into the preputial diverticulum called Balling up • Boar may show rectal copulation and leads to ejaculate : Pederasty

Objectives • Seminal evaluation and processing • Evaluation of bull fertility on the basis of its seminal characters • To make the Artificial insemination programme successful History Related to Semen Collection • The use of artificial vagina was started by Milovanov for semen collection. Before that method of placing the cotton ball in vagina was used. • AV for Dog was discovered by Amantea in 1914. Milovanov discovered AV for Bull, ram and stallion. Mckenzie discovered AV for boar (1931). • The electroejaculatory method of semen collection was initiated in a bull by Laplaud & Cassou and in ram by Gunn. • Burrows and Quinn (1937) developed the method of abdominal massage and pressure to collect semen in poultry.

• Mature sperm cells are undifferentiated cells which are the testicular product as well as indicators of testicular spermatogenic function. A spermiogram reflects the testicular activity and fertility status of a male animal. • Disease conditions, age, stress factors, vaccination or any altered physiological conditions may affect the seminal quality. Objectives of Semen Evaluation • To provide essential information about the fertility status of the male animal, though the fertilizing capacity of the animal cannot be completely defined by these tests. • To diagnose various pathological conditions of testes and male genitalia. • To determine the eligibility for further processing of semen cryopreservation • To determine the potential of semen for AI purposes

Semen • Semen, a fluid released from the male reproductive system during ejaculation, is made up of a liquid phase called seminal plasma and a cellular component called spermatozoa. • The term “spermatocrit” refers to the percentage of spermatozoa(v/v) in semen, which varies from less than 2% in pigs to more than 30% in sheep. It is about 10% in bull. Semen= Spermatozoa + seminal plasma Spermatozoon • In 1677, the scientist Van Leeuwenhoek first discovered the spermatozoa and called them ‘‘animalacula’’. • Spermatozoa or sperm cells or male gametes, develop within the wall of the seminiferous tubules as a result of a complex process known as spermatogenesis. • The spermatozoon is a highly specialized cell that performs the sole function of fertilizing an oocyte.

Sperm abnormalities may be genetical or morphological. They are directly related to the male infertility. • The wet mount method and phase contrast microscopy facilitate sperm visualization for abnormalities. Semen stained with vital stain can also be used for evaluating sperm abnormalities. Sperm morphology or related abnormalities can be evaluated by using eosin-nigrosine stain, carbolfuschin, aniline blue-eosine etc. • Generally, for head and tail abnormalities rose Bengal and William’s stain can be used while for acrosome and head abnormalities Giemsa stain can be used. Categorization of Sperm Abnormalities 1. On the basis of site of abnormality on the spermatozoa • Head abnormalities e.g. microcephalic head, macrocephalic/giant head, narrow or short sperm head, pyriform head, detached glea capitis (acrosomal cap), detached head and double head etc

• Semen cryopreservation – It is the process of preserving the semen in a suitable medium at an ultra-low temperature which reduces spermatozoa metabolism with the least production of metabolic waste products of sperm cells to prolong their life span. • Every fall of 100 C temperature decreases the metabolic rate of spermatozoa to half. Principle of various components involved in cryopreservation 1. Low temperature • Reduce metabolism (negligible) and least metabolic waste product • Decrease solute solubility leads to increased viscosity and physical resistance • Reduce motility • Prolong life span 2. Protection from cold shock and cryoinjury • When sperm cells are subjected to a reduction in temperature during the freezing process, there are: • Sperm membrane damages and lead to leakage of intracellular components like enzymes, ATP, K+ , cholesterol, lipoproteins etc. • Irreversible abnormality in motility pattern (bending of sperm tail)

Semen is needed to be extended with a suitable medium and cryopreserved without affecting its survivability and fertility for the purpose of inseminating female artificially. Qualities of Ideal Extenders • Semen extender is a nutrient media which provide energy to spermatozoa, protection from osmotic shock, thermal shock and various metabolic wastes for prolonging the life span of sperm cell with maintaining the acceptable fertilizing capacity. A semen extender should have qualities to protect spermatozoa during dilution and cryopreservation. These qualities may be : 1. Preserving sperm cell integrity (membrane, cellular, mitochondrial and acrosomal integrity etc) 2. Maintenance of osmotic pressure (isotonic; 280-300 mOsm/l) similar to seminal plasma 3. Providing nutritive elements as energy source for sperm metabolism (aerobic & anaerobic metabolism)

• After equilibration, a pre-freeze evaluation of semen is done. Pre-freeze motility should be more than 70% at 370 C which makes the semen eligible for freezing. Semen Packaging • Semen is packaged in suitable container like pellets, glass ampoules, tupol (polyethene tubes) and semen straws. • In the Ampoule method, semen is packed in glass ampoules and stored in LN2 . Here labeling and handling is easier with less chances of contamination. But more storage space, more time taking to thaw and the use of glass catheter for insemination make their the use less convenient. • In Pellet methods, diluted semen is frozen by pouring it into the holes (0.1, 0.2 ml) made on dry ice (Solid CO2 at 790 c) and these pellets (frozen semen balls) are stored in LN2 . Here glycerolization is not needed as well as process is also quick but labeling is difficult and the chances to get contaminated are more. Handling pellets for insemination is also difficult (breakage or getting stuck to forceps during retrieval).

• Artificial Insemination (AI) is the technique of deposition of semen (Fresh, Diluted or frozen thawed) in the female genital tract by artificial means for the purpose of conception. • It is first generation biotechnology technique. It is the most economical and best technique which has been widely accepted for genetic improvement and production (calf crop and milk production) in farm animals. • All the aspects like selection of bull, semen collection, semen evaluation, processing of semen, freezing, cryopreservation, thawing and insemination are equally important for making AI successful.History Related To AI • In the fourteenth century an Arabian placed a cotton ball in the mare's vagina and collect it after mating (soaked in semen) with a high-grade stallion; then by inserting this cotton ball into the vagina of desired mare and got it conceived. • Anton Van Leeuwenhoek (Dutch scientist, 1678) and his assistant, Hamm, discovered sperm cell and called it as “Animalcule”.

There are some methods to control male reproductive behaviour for better breeding and managemental practices i.e. Vasectomy and Caudal epididymectomy, Penile surgeries, and castration etc. • Surgical and non surgical interventions in the male reproductive system are carried out for: • To make teaser bull • To make bull sterile • To prevent the transmission of venereal diseases • Population control or Breeding management • To rectify any ailment or deformity Teaser Bull • A male animal whose capability of spermatogenesis as well as libido is maintained but it is not able to impregnate a female. • For the preparation of teaser bull or Gomer bull, Male animal should be selected well before the start of breeding season. Bull should be young vigorous and fertile. It should be of moderate adult size with mild temperament. Heavy and large sized bull is tough to handle, needs more time for surgery, has chances of more hemorrhage, and may have more post surgical complications.