Introduction
Artificial insemination (AI) is considered a valuable tool for the poultry industry (Benoff et al., Reference BENOFF, ROWE, FUGUAY, RENDEN and SCOTT1981) due to the efficient utilisation of males, which is not possible under natural mating. This decreases the cost of poultry production directly by reducing the number of cockerels needed for male gamete production (Benoff et al., Reference BENOFF, ROWE, FUGUAY, RENDEN and SCOTT1981). AI was the first biotechnological tool applied to increase poultry production, as it allowed wider use of genetically superior cockerels with high productive performance (Benoff et al., Reference BENOFF, ROWE, FUGUAY, RENDEN and SCOTT1981). Pioneering efforts to establish AI as a practical procedure began in Russia in 1899 by Ivanow, who studied AI in domestic farm animals including poultry (Ivanow, Reference IVANOW1922). This technique was used successfully for the first time in birds almost a century ago, when Ivanov produced fertile chicken eggs using semen recovered from the ductus deferens of a cockerel (Ivanow, Reference IVANOW1907). In chickens, artificial semen collection was made easier by devices developed by Ishikawa (Reference ISHIKAWA1930). In 1935, Burrows and Quinn reported an abdominal massage technique for chickens, which was improved and referred to as ‘milking the male’ (Burrows and Quinn, Reference BURROWS and QUINN1937). The technique for semen collection followed by AI was developed by Quinn and Burrows (Reference QUINN and BURROWS1936). Hence, these researchers were considered the fathers of avian AI, and their basic method is still being employed in various poultry species.
AI was widely used with the introduction of laying cages in Israel (Thumin, Reference THUMIN1951) and Australia (Skaller, Reference SKALLER1951). In USA, AI was used to improve the fertility of broilers in the same period (Moultrie, Reference MOULTRIE1956). In India, this technique was adopted in poultry with the introduction of the All India Coordinated Research Project on Poultry in 1975. Usually, AI is extensively used with freshly collected semen due to its ease of collection and the proximity of hens in large breeding farms for insemination. Since the 1960s, AI technique has emerged as the most critical component of reproduction in turkeys and it is used almost exclusively for commercial flock production. The differences in the size of toms (large white strains weigh approximately 33 kg) and hens (approximately 9 kg at the onset of lay) results in unsuccessful natural mating and consequent low fertilisation of heavy, broad-breasted strains which has forced the adoption of AI in commercial production (Lake, Reference LAKE1983; Donoghue and Wishart, Reference DONOGHUE and WISHART2000).
AI is gaining momentum in other poultry species. For example, as fertility in broiler breeds continues to decline as males are selected for growth, AI may become cost-effective in broiler breeder management (Reddy, Reference REDDY1995). Due to the presence of cloacal gland foam and low semen volume in quail (Mohan et al., Reference MOHAN, MOUDGAL, SASTRY and SINGH2002; Shit et al., Reference SHIT, SINGH, SASTRY, PANDEY, MOHAN and MOUDGAL2010) and guinea fowls (Mohan et al., Reference MOHAN, KHANDAY, SINGH and TYAGI2013), AI is not easy in these species. Similarly, AI is not simple in ducks and geese, because, unlike chickens and turkey hens, the oviduct cannot be everted and so commercial demand for AI in these species is limited (Cooper, Reference COOPER1977). AI techniques have been successfully adapted to cranes and other wild birds (Gee, Reference GEE1995; Blanco et al., Reference BLANCO, WILDT, HOLFE, VOELKER and DONOGHUE2009), which is important in preserving endangered species, to assist in creating viable and self-sustaining populations. Out of approximately 10,000 known avian species, 13% are considered as threatened. This means that nearly 1,375 species are at the serious risk of extinction (International Union for Conservation of Nature and Resources, 2018).
The main objective of this review is to discuss the history and current status of AI techniques for economic poultry production and to protect critically endangered avian species. The components of successful AI, including semen collection, evaluation, dilution, storage, insemination of hens, timing of insemination, storage temperature, sperm doses/insemination and frequency of semen collection and insemination are discussed along with the significance of this technique over natural mating.
Semen collection
To successfully perform AI, a clean semen sample of sufficient volume is required regularly. The old method of semen collection allowed the cockerel to mate with hen, and then hens were killed and semen was surgically collected from the oviduct. This drastic method forced the development of the current techniques, termed ‘abdominal massage method’, as described by Burrows and Quinn (Reference BURROWS and QUINN1937). This involves two persons and is common for collection from chickens and turkeys, as well as in pigeons (Klimowicz et al., Reference KLIMOWICZ, LUKASZEWICZ and DUBIEL2005). The erection of the copulatory organ and the ejaculation reflex in turkeys, domestic fowls, guinea fowls (Lake and Stewart, Reference LAKE and STEWART1978), quails (Marks and Lepore, Reference MARKS and LEPORE1965) and pheasants (Spiller et al., Reference SPILLER, GRAHAME and WISE1977) occurs simultaneously in response to massage and the major portion of the spermatozoa are obtained in the first ejaculate. Adaptations of the massage method have been made for other avian species like waterfowls (drakes) which have penis-like copulatory appendages (Nishiyama et al., Reference NISHIYAMA, NAKASHIMA and FUJIHARA1976). In Japanese quail, the technique of semen collection was applied by Shit et al. (Reference SHIT, SINGH, SASTRY, PANDEY, MOHAN and MOUDGAL2010) and required the removal of foamy substance from the cloacal gland before semen collection by massaging. In ratites, semen of good quality can be collected by using the artificial cloaca (Malecki et al., Reference MALECKI, RYBNIK and MARTIN2008) instead of the manual massage method that is labour intensive and unreliable (Hemberger et al., Reference HEMBERGER, HOSPES and BOSTEDT2001). Various methods of semen collection in ostriches have been described by Rozenboim et al. (Reference ROZENBOIM, NAVOT, SNAPIR, ROSENSHTRAUCH, EL HALAWANI, GVARYAHU and DEGEN2003).
Several factors viz. age, season, lighting schedule, body weight, nutrition, management and spermatogenesis influence semen collection, quality and fertility of birds (Maule, Reference MAULE1962; Sturkie, Reference STURKIE1986; and Mohan et al., Reference MOHAN, SHARMA, KOLLURI, SINGH, TYAGI and KATARIA2016). In addition, endocrine disruptors particularly bisphenol-A, have been shown to compromise sperm quality (Singh et al., Reference SINGH, SHAFEEQUE, SHARMA, . PANDEY, SINGH, MOHAN, KOLLURI, SAXENA, SHARMA, SASTRY, KATARIA and AZEEZ2015; Reference SINGH, SHAFEEQUE, SHARMA, SINGH, MAHARAJAN, SINGH, SASTRY, SAXENA, MOHAN and AZEEZ2016). Semen collection from cockerels may be influenced by handling of the birds and the training of the operator (Cooper, Reference COOPER1965). Semen can be obtained from White Leghorn (WLH) chickens as early as 16 weeks (Lorenz and Lerner, Reference LORENZ and LERNER1946) and from turkeys at 28 weeks (Carson et al., Reference CARSON, LORENZ and ASMUNDSON1955), but semen with high fertilising ability can be obtained from 26-28-week-old chickens and 32-36-week-old turkeys. A higher volume of semen can be obtained in the afternoon and this timing matches the mating behaviour of birds (Lake and Wood-Gush, Reference LAKE and WOOD-GUSH1956). The average volume of semen per collection ranges from 0.05-0.50 ml in light breeds of chicken and from 0.1-0. 9 ml in heavy breed males (Lake and Stewart, Reference LAKE and STEWART1978). In light-weight turkeys, the semen volume is 0.08-0.30 ml, whereas in heavy-weight males it is 0.1-0.33 ml (Lake and Stewart, Reference LAKE and STEWART1978). The fertilising ability of the spermatozoa of chickens up to three years old is the same; however, volume decreases with age. Thus, it could be beneficial to keep a valuable male for several seasons to increase poultry production (Cooper, Reference COOPER1965). Male birds need to be routinely trained for semen collection for several days prior to the actual date of AI application to guarantee good quality of semen. As the bird's phallus is located very close to the anus in the cloacal region, it enhances the probability of faecal contamination of the semen. Therefore, to collect clean semen samples, feed should be withdrawn 12 h prior to semen collection.
frequency of semen collection
The rate at which semen is collected determines the success of AI, and the number of good quality spermatozoa is an important factor in determining fertility level (Tabatabaei, Reference TABATABAEI2010; Beulah, Reference BEULAH2017). It is important to note that the proportion of naturally degenerating spermatozoa in the vas deferens increases with long periods between semen collections. This is because spermatozoa are reabsorbed by certain lining cells, a process termed ‘vasoligation’ (Tingari and Lake, Reference TINGARI and LAKE1972). Thus, for a consistent supply of good quality semen during the breeding season, semen should be collected from males at a particular frequency, although this varies among breeds and species. A thrice weekly frequency (alternate days) maintained the optimum output of spermatozoa, resulting in good fertility in chickens (Lake and Stewart, Reference LAKE and STEWART1978), while in Pekin breeder ducks two collections per week were shown to yield better results (Nahak et al., Reference NAHAK, GIRI, MOHANTY, MISHRA and DASH2015).
Evaluation of semen quality
In poultry, the quality of semen is the prominent determinant of fertility and hatchability of eggs (Peters et al., Reference PETERS, OMIDIJI, IKEOBI, OZOJE and ADEBAMBO2004) and is the major basis of profitability of poultry enterprises. There are two major reasons for the evaluation of semen quality, firstly, to ensure that only males producing semen of good quality are kept on the farm and secondly, to measure the concentration of spermatozoa and the volume of semen. This allows calculations for the appropriate dilution to be made to obtain 80-100 million spermatozoa per insemination dose. In 1960s and 1970s, inseminations were based on the semen volume per dose; in the 1970s and 1980s, inseminations were based on the number of sperm cells per dose; and in the late 1980s and in 1990s, inseminations were based on the number of viable sperm per dose (Van Wambeke and Huyghebaert, Reference VAN WAMBEKE and HUYGHEBAERT1989). Determining sperm concentration has several advantages: it provides a basis for calculating the number of sperm per insemination dose and serves as a measure of semen quality (Bakst, Reference BAKST1993; Senger, Reference SENGER2003). Semen collected from domestic cockerels contains an average sperm concentration of 5 billion sperm cells/ml (Gordon, Reference GORDON2005), whereas Hafez and Hafez (Reference HAFEZ and HAFEZ2000) reported 3-7 billion sperm cells/ml. Sperm motility is an indicator of live sperm and of the quality of the semen sample. Evaluation of sperm motility is conducted with fresh and diluted semen and generally analysed under the light microscope (100× magnification) (Hafez and Hafez, Reference HAFEZ and HAFEZ2000). Sperm motility is a primary determinant of fertility in domestic fowls (Donoghue et al., Reference DONOGHUE, HOLSBERGER, EVENSON and FROMAN1998). However, visual examination of semen cannot be ignored (Peters et al., Reference PETERS, SHOYEBO, LLORI, OZOJE, IKEOBI and ADEBAMBO2008). A semen sample of good quality is thick and pearly white in colour (Cole and Cupps, Reference COLE and CUPPS1977), and any other colour is an indicator of contamination, e.g. yellow and green-coloured semen is indicative of faecal or urine contamination (Lake, Reference LAKE1983). A brownish red pigment or reddish colour demonstrates the presence of blood (Etches, Reference ETCHES1996). However, the semen of domestic fowl ranges from a dense opaque suspension to a watery/transparent fluid (Hafez, Reference HAFEZ1978; Mohan and Moudgal, Reference MOHAN and MOUDGAL1996). An excess of transparent fluid in semen can be detrimental to spermatozoa, as it is rich in calcium ions which causes clumping (Mohan and Moudgal, Reference MOHAN and MOUDGAL1996).
The morphology of spermatozoa can serve as a tool to evaluate semen quality. Blesbois (Reference BLESBOIS2007) described an eosin-nigrosin staining technique to examine the morphology of cockerel semen. Only morphologically normal spermatozoa are capable of ascending through the vagina of the hen to the sperm storage tubules (SSTs; Bakst et al., Reference BAKST, WISHART and BRILLARD1994). Several physical and biochemical tests are also available for the evaluation of semen (Peters et al., Reference PETERS, SHOYEBO, LLORI, OZOJE, IKEOBI and ADEBAMBO2008; Biswas et al., Reference BISWAS, MOHAN and SASTRY2009; Mohan et al., Reference MOHAN, SINGH, SASTRY, MOUDGAL, SHIT and BISWAS2011; Reference MOHAN, KHANDAY, SINGH and TYAGI2013). Semen evaluation is traditionally conducted by a laboratory technician looking through a microscope and manually counting sperm, progressive motility (usually given a score ranging from 1 (poor) to 4 (best)), and morphology of spermatozoa (damage and defects). These observations were completely subjective. To overcome such problems, a sophisticated electronic imaging system to visualize sperm and associated with an advanced software program to evaluate large number of individual sperm parameters has been developed. This is usually referred to as computer assisted semen analysis (CASA). More importantly, semen analysis by CASA is accurate and reproducible that would be very helpful for prediction of fertility potential of individual cockerel. Initially, CASA has been standardised for semen analysis of different mammalian species where spermatozoa have spherical and short head. However, this system cannot be exploited for morphological analysis of avian spermatozoa which are filiform in shape with elongated and narrow heads (Santiago-Moreno et al., Reference SANTIAGO-MORENO, ESTESO, VILLAVERDE-MORCILLO, TOLEDANO-DÍAZ, CASTAÑO, GIMENO MARTÍNEZ, VELÁZQUEZ, LÓPEZ-SEBASTIÁN and LÓPEZ GOYA2016). Villaverde-Morcillo et al. (Reference VILLAVERDE-MORCILLO, ESTESO, CASTAÑO, TOLEDANO DÍAZ, LÓPEZ-SEBASTIÁN, CAMPO and SANTIAGO-MORENO2015) analysed avian sperm head characteristics of cockerel and partridge semen using computer assisted light microscopy. In chicken, sperm motility and concentration were usually assayed as the method described by Santiago-Moreno et al. (Reference SANTIAGO-MORENO, CASTANO, TOLEDANO-DIAZ, COLOMA, LOPEZ-SEBASTIAN, PRIETO and CAMPO2012) using the CASA system coupled to a phase contrast microscope (Nikon Eclipse model 50i; negative contrast) and employing Sperm Class Analyzer (SCA, Barcelona, Spain) v.4.0 software (Microptic S.L., Barcelona, Spain). Following the same procedure in rooster, Davila et al. (Reference DAVILA, MUÑOZ, TAPIA, FERRUSOLA, DA SILVA and PEÑA2015) recorded the percentage of motile spermatozoa, immotile sperm, non-progressive motility (sperm moving their tails but not moving forward, or swimming slowly in a curve) and progressive motility (sperm swimming quickly forward in a straight line). Sperm movement characteristics, such as straight-line velocity (VSL), curvilinear velocity (VCL), and average path velocity (VAP) were also analysed by CASA. According to their general velocity, spermatozoa were classified as slow (<10 μm/sec), medium (10-50 μm/sec) or rapid (>50 μm/sec).
Sayed et al. (Reference SAYED, ABOUELEZZ, AMIRA and ABDEL-WAHAB2017) assessed sperm motility and swimming velocity in Egyptian indigenous chicken using a new CASA plugin system. A mechanised sperm quality analyser (SQA) can be used as a practical, inexpensive platform for quality analysis to generate sperm quality index (SQI) values. This index is the reflection of sperm movement or activity in the given semen sample (McDaniel et al., Reference MCDANIEL, HANNAH, PARKER, SMITH, SCHULTZ and ZUMWALT1998a) and considers progressive motility, concentration and normal morphology. As interpreted from SQA, SQI refers to a single consolidated number that provides an estimate of overall sperm quality and quantity in avian semen (Wishart and Wilson, Reference WISHART and WILSON1997; McDaniel et al., Reference MCDANIEL, HANNAH, PARKER, SMITH, SCHULTZ and ZUMWALT1998a; Reference MCDANIEL, PARKER, YEATMAN, SCHULTZ and ZUMWALT1998b). Akashi et al. (Reference AKASHI, MIZUNO, OKUMURA and FUSE2005) observed a high correlation for sperm concentration, motility and morphology by employing the three methods of semen analysis viz. SQA, CASA and manual analysis.
Biochemical examination indicated a lower superoxide dismutase activity in turkey semen than in chicken semen suggesting that the former is more susceptible to oxygen toxicity during in vitro storage (Froman and Thurston, Reference FROMAN and THURSTON1984). Brown et al. (1971) examined enzymes in spermatozoa and selected the release of glutamic oxaloacetic transaminase (GOT) as the best indicator of cell damage. Recently, RNA transcripts have been developed as potential biomarkers of male fertility of mammalian species including chicken, and a protocol was standardised for the isolation and purification of RNA from chicken spermatozoa (Shafeeque et al., Reference SHAFEEQUE, SINGH, SHARMA, MOHAN, SASTRY, KOLLURI, SAXENA, TYGAI, KATARIA and AZEEZ2014). The fertilising quality of spermatozoa can be determined directly from the population available in the peri-vitelline membrane (Wishart, Reference WISHART1987), or indirectly by counting the number of holes created by the hydrolysis of the inner peri-vitelline layer (Wishart and Wood, Reference WISHART and WOOD1994).
Semen diluent and storage temperature
Chicken semen is highly concentrated, viscous and often low in volume and degradation of avian sperm occurs relatively quickly if left undiluted (Lake and Stewart, Reference LAKE and STEWART1978). Semen diluents are the buffered salt solutions designed to ‘extend’ the volume of semen, which provides an optimum environment to ensure viability, increasing the number of insemination doses from each collection (Vasicek et al., Reference VASICEK, KUZELOVA, KULIKOVA and CHRENEK2015) and ensuring uniform distribution of spermatozoa in diluents. The composition of semen diluents is based on the biochemical attributes of poultry semen (Lake, Reference LAKE1995) and provides osmotic pressure (330-400 mOsm) and pH (7.0-7.4) identical with seminal plasma (Thurston Reference THURSTON1995; Miškeje et al., Reference MIŠKEJE, SLANINA, PETROVIČOVÁ and MASSÁNYI2013). The development of diluents initially began with the simple use of NaCl solutions followed by complex diluents containing osmotic regulators, energy substrates and buffers (Bootwalla and Miles, Reference BOOTWALLA and MILES1992). Some of the notable diluents for 24 h storage are the Lake diluent (Lake, Reference LAKE1960), Beltsvillie Poultry Semen Extender (BPSE) by Sexton (Reference SEXTON1977), EK extender (Łukaszewicz, Reference ŁUKASZEWICZ2002) and Tselutin extender (Tselutin et al., Reference TSELUTIN, NARUBINA, MAVRODINA and TUR1995). Despite their success for use in the poultry industry, these diluents have been reported to cause a wide range of variations in fertility. The recently developed the ‘CARI’ poultry semen diluent (Mohan et al., Reference MOHAN, SASTRY and KATARIA2017b) has been shown to give better fertility than others in various poultry species. As well as these products, commercial diluents like Glutac (IMV Technologies, France) and Lago avian semen extender (Hygieia Biological Laboratories, California) have been largely used for avian AI.
Generally, low temperature (5°C or lower) has been used for the storage of semen. This seems logical in view of the lowered metabolism of spermatozoa at this temperature, and a refrigerator temperature of 2-5°C is attractive for practical reasons (Sexton, Reference SEXTON1977; Van Wambeke, Reference VAN WAMBEKE1978). Brillard (Reference BRILLARD2009) stated that, for long storage periods, semen should be subjected to low storage temperatures, ranging from 4-10°C. However, a lower temperature particularly 0°C should be avoided. Donoghue and Wishart (Reference DONOGHUE and WISHART2000) advocated that, for good fertility of stored semen in vitro, samples have to be preserved at 2-8°C. Slanina et al. (Reference SLANINA, PETROVIČOVÁ, MIŠKEJE, KŇÍŽAT, MIRDA, LUKÁČ, TRANDŽÍK, PETROVIČOVÁ and MASSÁNYI2015) found that turkey sperm can be stored at 4-8°C. Mohan et al. (Reference MOHAN, SHARMA, KOLLURI, GOPI, TYAGI and KATARIA2017a; Reference MOHAN, SASTRY and KATARIA2017b; Reference MOHAN, SHARMA, KOLLURI, GOPI, TYAGI and SINGH2017c) stored chicken semen for 24 h at 7-8°C with very good fertility.
Dilution rate of semen
The dilution rate and the type of diluent have a profound effect on sperm motility, ionic balance, gas exchange and ATP (energy) utilisation (Parker and McDaniel, Reference PARKER and MCDANIEL2006). A two to three-fold dilution (moderate dilution) is considered to be the optimum for in vitro storage of chicken semen. A high dilution rate (above five-fold) shortens the life span of spermatozoa by stimulating sperm motility and metabolic activity (Sexton, Reference SEXTON1979; Parker and McDaniel, Reference PARKER and MCDANIEL2006). In contrast, semen diluted at a low level (below 1:1) does not survive at low temperatures (Sexton, Reference SEXTON1979). Recently, Mohan et al. (Reference MOHAN, SHARMA, KOLLURI, GOPI, TYAGI and SINGH2017c) preserved chicken (WLH) semen in CARI poultry semen diluent for 24 h in different dilutions, i.e. 1:1, 1:2, 1:3, 1:4 and 1:5. Superior fertility (91.07±1.91%) was observed at a dilution rate of 1:2 (89.10×106 sperm per dose) whereas inferior fertility noticed with 1:1 (76.93±2.54%) and 1:5 (49.40±2.54%) rates during the fertile period of up to five days after AI. Beulah (Reference BEULAH2017) used freshly ejaculate diluted chicken semen in CARI diluent with dilutions of 1:2 to 1:20 under the same duration of fertile period and saw a fertility higher than 90% in up to 1:8 dilution (29.70×106 sperm per insemination dose), whereas a fertility of 77% was observed at 1:20 (12.71×106 sperm per AI dose).
Avian semen is highly concentrated; thus, in a low dilution, normal cells remain in close association with deformed cells which are the normal constitutes of any cell population. Morphologically abnormal cells could mechanically injure normal spermatozoa by mutual contact (Nei, Reference NEI1981). Therefore, a low dilution rate of avian semen is not practical. These variations at low and high dilutions are attributed to the dilution effect (Mann, Reference MANN1964). A dilution rate of up to 1:10 was able to produce higher fertility and hatchability provided the inseminating dose of sperm was at least 100×106 spermatozoa (Bratte and Ibe, Reference BRATTE and IBE1989; Sexton, Reference SEXTON1979). However, this mechanism needs further research. Any excessive dilution must be avoided since the number of spermatozoa/insemination dose will be reduced.
Artificial insemination of hens
During natural copulation in fowls, semen is usually deposited in the shallow position in the vagina of the hen. During AI, it is necessary to evert the distal part of oviduct (vagina) and deposit the semen to a depth of 2-4 cm (or as close as possible to the sperm host gland) for optimum fertility of eggs laid daily in succession by hens over the following week. Usually, chicken semen is inseminated at the depth of 2-3 cm in the vagina (Artemenko and Tereshchenko, Reference ARTEMENKO and TERESHCHENKO1992). Sometimes insemination can be made at the depth of 3-4 cm (Kurbatov et al., Reference KURBATOV, NARUBINA, BUBLAYEVA and TSELUTIN1984). Insemination to the depth of 5-6 cm in chicken requires penetration of the uterus with the syringe. This type of insemination is detrimental to the health of birds and reduces fertility (Artemenko and Tereshchenko, Reference ARTEMENKO and TERESHCHENKO1992).
After obtaining good semen samples, the actual insemination of the hen can be performed by two persons (Quinn and Burrows, Reference QUINN and BURROWS1936). One person applies the proper pressure on the left side of the abdomen so that the hen everts (turns inside out) her vaginal orifice through the cloaca. This procedure is termed ‘cracking’, ‘venting’ or ‘everting’ the hen. At the same time, the semen is deposited by the second person to a depth of 2-4 cm into the vaginal orifice concurrently with the withdrawal of pressure on the hen's abdomen. Insemination can be done with sterile straws, syringes or plastic tubes. In large scale, commercial operations, automated semen dispensers using individual straws loaded with a set volume with the desired sperm concentration are commonly used. If hens are accustomed to egg production, it is relatively easy to evert the oviduct. The depth of insemination depends upon the species of the bird and length of the vagina. Avian spermatozoa are normally inseminated into the vagina from where less than 1% of the sperm enters in to the SSTs located at the utero-vaginal junction (Bakst et al., Reference BAKST, WISHART and BRILLARD1994). The sperm is stored here for several days or weeks to participate in the subsequent process of fertilisation. According to Hafez and Hafez (Reference HAFEZ and HAFEZ2000), in mammals the sperm spends a relatively short time in the female tract while in chickens and turkeys, the sperm can spend a much longer period in the oviduct before fertilising the egg yolk cell (up to 32 days in chickens and 70 days in turkeys). From the SSTs, spermatozoa will migrate towards the upper part of oviduct to a second storage site (sperm nests) situated at the junction of the magnum and infundibulum (Aisha and Zain, Reference AISHA and ZAIN2010). The passage of an ovum into the infundibulum stimulates the release of spermatozoa from sperm nests and fertilisation takes place (Aisha and Zain, Reference AISHA and ZAIN2010).
timing of AI
The time of the day at which insemination is performed, both for artificially or naturally mated chickens, plays a significant role in fertility. In the evening, the incidence of the hard-shelled eggs in the uterus of the hens is rare. Hence, inseminations performed in the afternoon or evening yield higher fertility than those performed in the morning (Christensen and Johnston, Reference CHRISTENSEN and JOHNSTON1977; Aisha and Zain, Reference AISHA and ZAIN2010). The presence of hard-shelled eggs in the uterus of the hens at or near the time of AI causes a decrease in the level of fertility. Most of the spermatozoa inseminated in chickens and turkeys within 1-3 h prior to or just after oviposition are eliminated by the contraction of the vagina involved in the process of oviposition (Brillard and Bakst, Reference BRILLARD and BAKST1990). As far as ratites are concerned, inseminating (400 million spermatozoa per dose) females on the day following oviposition during the morning (09:00 to 10:00 AM) resulted in longer lasting (two weeks) of fertile period (Malecki and Martin, Reference MALECKI and MARTIN2004; Malecki et al., Reference MALECKI, RYBNIK and MARTIN2008). However, optimum timing of AI remains undetermined (Malecki et al., Reference MALECKI, RYBNIK and MARTIN2008).
doses of sperm and frequency of insemination
To maintain a constant high level of fertility throughout a breeding season, a minimum number of good quality spermatozoa must be inseminated at regular intervals. The interval depends upon the type of bird but is generally about one week under farm conditions. If the interval between inseminations is extended beyond one week, fertility declines significantly (Sexton, Reference SEXTON1977). The hatchability of eggs is reduced due to a greater incidence of infertile eggs and gradual increase in early embryonic mortality. The latter may be due to ageing spermatozoa in the oviduct (Lodge et al., 1971; Reference LODGE, AX and FECHHEIMER1974). Tabatabaei (Reference TABATABAEI2010) reported 88% fertility in chicken inseminated with 100 million spermatozoa, whereas Beulah (Reference BEULAH2017) revealed 94% fertility in WLH using 89 million spermatozoa. In native breeds of chicken (e.g. naked neck) Mohan et al. (2018, personal communication) found 89% fertility using 80 million spermatozoa. No advantage was gained by inseminating more than 100 million fresh, good quality spermatozoa into a female at any one time (Lake, Reference LAKE1983). When the semen is stored for 6 h at low temperature, 125-150 million spermatozoa should be inseminated per hen per week (Van Wambeke, Reference VAN WAMBEKE1987). Approximately 200-250 million spermatozoa were required to produce high levels of fertility with fowl semen stored after 24 h at 2-5°C (Van Wambeke, Reference VAN WAMBEKE1978; Reference VAN WAMBEKE1987). Mohan et al. (Reference MOHAN, SHARMA, KOLLURI, GOPI, TYAGI and SINGH2017c) achieved high fertility (91.07±1.91%) after 24 h storage of chicken semen by inseminating 89.10×106 sperm per AI dose. Deep-frozen semen requires a higher number (250 million) of spermatozoa/insemination to insure a great security margin for fertility (Van Wambeke, Reference VAN WAMBEKE1986).
In domestic fowl, the age of the bird, sperm numbers and types of hens (broiler or layer) may affect the fertility of eggs (Talebi et al., Reference TALEBI, BATAVANI and TABATABAEI2009). Douard et al. (Reference DOUARD, HERMIER, MAGISTRINI and BLESBOIS2003) reported a significant effect of age on semen quality, which was visible at 47 weeks of age and corresponded to a reduction of 15% in viability. At the age of 52 weeks, a 20% loss in motility was seen for turkey spermatozoa. These researchers showed higher fertility rates when AI was performed during 32-35 and 39-42 weeks in comparison to 44-47 weeks of age in males (93.90 and 97.50% vs. 81.80% fertility respectively). Loss of semen quality was observed with the advancement of male age (44-52 weeks). Slanina et al. (Reference SLANINA, PETROVIČOVÁ, MIŠKEJE, KŇÍŽAT, MIRDA, LUKÁČ, TRANDŽÍK, PETROVIČOVÁ and MASSÁNYI2015) reported better semen quality in turkey males collected at the age of 35-42 weeks than at 63-73 weeks. De Reviers and Brillard (Reference DE REVIERS and BRILLARD1984) reported that 49-52-week-old hens required more number of spermatozoa than 28 to 31 weeks old hens to achieve maximum fertility. Brillard and McDaniel (Reference BRILLARD and McDANIEL1986) found high fertility (93.3%) in young hens inseminated at weekly intervals with moderate doses (125 million) of sperm, whereas, in old hens, high sperm doses (250 million) were inadequate to maintain fertility at a similar level to young hens. This suggested more sperm loss in sperm host glands in old hens than young.
Joyner et al. (Reference JOYNER, REDDIE and TAYLOR1987) discussed the reduction of egg production in domestic hens with increasing the age as being associated with high incidence of thin shelled and cracked eggs. Older hens produced fewer eggs but of a larger size than younger ones. Age significantly affected the rate of lay in chicken (75.4% for young vs. 62.6% for old) with sequence length of 7.7 days and 2.6 days for young and old hens respectively. Laying hens are characterised by greater number of eggs laid (84.1% rate of lay) in larger sequences than with broiler breeders (54% rate of lay) (Tumova and Gous, Reference TUMOVA and GOUS2012).
significance of AI over natural mating
Using semen diluent, the services of superior male can be used maximally by AI, which is not possible under natural mating. From an economic perspective, diluents reduce the number of males required for AI hence, saving feed cost due to having less numbers of males and saving space, maintenance and operating costs. Natural mating fixes an upper limit of males to females at a ratio of about 1:10, whereas AI allows breeders to service 100 hens from a single male. This increased the use of males, combined with the ability to recognise hens not fit for laying eggs (10-30%) in the flock during the AI process, provides an excellent means to reduce (at least by 20%) the cost (Hammerstedt, Reference HAMMERSTEDT1996). Recently, Mohan and Sharma (Reference MOHAN and SHARMA2017) advocated that fresh semen (5.34 x 109 sperm/ml) obtained from a cockerel (0.5 ml) can be inseminated in to 70 hens/day after dilution with CARI diluent (1:6) giving 38 million sperm per dose for AI (0.05 ml diluted semen). Nearly 90% or above fertility can be achieved in this manner. Using a single male on alternate days (four times in a week), 280 hens could be covered, whereas, under natural mating, the cockerel to hen ratio of 1:8 was kept.
AI in avian species results in better fertility than natural mating (Saeki and Nagomi, Reference SAEKI and NAGOMI1964; Brillard, Reference BRILLARD2003; Mohan et al., Reference MOHAN, SHARMA, KOLLURI, SINGH, TYAGI and KATARIA2016). Even though natural mating can lead to good fertility, rates can be enhanced further simply by adding AI to the reproductive programme (Gee et al., Reference GEE, BERTSCHINGER, DONOGHUE, BLANCO and SOLEY2004). Advantages from overall fertilisation rate and hatchability results in the reduction of the cost per unit of day-old chicks hatched (Brillard, Reference BRILLARD2003). Similar advantages in terms of reproductive success can be expected if avian AI techniques are extended into endangered birds.
Conclusions
The development of AI technology has resulted significant improvements in poultry production. However, this technique needs to be developed for non-domesticated birds which are at the serious risk of extinction, and more research is required to facilitate this. Reproductive performance in domesticated birds require more research to ascertain issues affected by disease, age, nutrition, photoperiod and other managemental factors. Non-laying hens should be recognised via AI procedures during the period of maximum flock production and be marked for removal. Various factors that reduce the semen quality due to improper handling, processing and storage need to be identified and tackled. To improve the semen quality during storage, removal of damaged cells and concentrates from viable spermatozoa should be undertaken. This may augment the fertilising ability of live spermatozoa. Going forward, traditional semen evaluation methods should be strengthened with molecular techniques. New molecular markers linked with high fertility and hatchability need to be investigated in poultry to select the male soon after hatching, rather than waiting for the onset of semen production.
Conflict of interest
The authors do not have conflict of interest in this work.
