In physiological condition, avian sperm can be stored within the sperm storage tubules of female reproductive tract and may able to fertilize eggs up to 15 weeks. The long-term viability and fertilizing ability of sperm is reduced when avian sperm are stored in vitro conditions. The motility, viability and fertilizing ability of avian sperm depends on in vitro storage conditions. Many factors can affect in vitro sperm motility, viability and fertilizing ability such as storage temperature, pH of extenders, osmolarity, sperm dilution rate, and seminal plasma. Researchers are trying to extend longevity of avian sperm during in vitro condition by applying the knowledge of in vivo sperm storage mechanism(s) and sperm biology. This paper reviews the sperm motility, viability and fertilizing ability of main poultry species stored in vitro conditions. This study will help to understand a scenario of in vitro avian sperm motility, viability and their fertilizing ability.
In physiological condition, the capacity of sperm to store in the female reproductive tract is relatively commonplace in reptiles, fishes, birds and amphibians (Holt, 2011). In mammals, some species of bat can also store sperm in the female reproductive tract for several months (Holt and Lloyd, 2010). Most mammalian females possesses a specialized reproductive structure for sperm storage in vivo condition (Suarez, 2010). Depending on the species, the capacity of sperm storage period and fertilizing ability varies from several hours to years. In avian species, ejaculated sperm can be stored in the lumen of sperm storage tubules (SSTs) and may retaining fertilizing ability up to 15 weeks at body temperature 41 °C (Sasanami et al., 2013). The process how sperm remains in the female reproductive tract for longer period of time still not fully understood. We have limited information regarding long-term sperm storage regulatory mechanism in vivo condition. The researchers are eagerly trying to discover the mechanisms underlying long-term sperm storage in vivo condition. Research previously conducted by Matsuzaki et al. (2015) found that sperm remains in the lumen of SSTs in quiescent state of motility at low pH (<6.0) under hypoxic condition. This unique feature may prolong sperm storage in the SSTs and enables female poultry to produce a series of fertile eggs following a single copulation event or artificial insemination (AI).
Except cryopreservation, the biotechnologists have not yet been able to develop any artificial method of achieving the goal of long-term sperm viability in vitro condition. In contrast to domestic livestock species, cryopreservation is not a reliable source for storing avian germplasm due to low fertilizing ability of frozen/thawed sperm (Long, 2006; Long et al., 2014). Unlike mammals, the avian sperm is probably not a reliable source due to differences in sperm shape, membrane fluidity and high amount of polyunsaturated fatty acids present in avian sperm (Çiftci and Aygün, 2018). Due to cryopreservation, the plasmalemma of avian sperm damages and spermatozoa becomes abnormal (Bakst and Sexton, 1979). Cryopreservation damages avian sperm, decreases sperm survivability and morphological integrity which is a major limitation of avian sperm storage (Blesbois et al., 2005). As avian sperm can be stored in the reproductive tract for a considerable period of time, this fact has been a source of interest and researchers are attempting to improve in vitro sperm survivability by applying the knowledge of in vivo sperm survival mechanisms.
The goal of modern poultry industry is to provide high quality meat and eggs. Nowadays, artificial insemination (AI) is being used widely in the breeding farm to propagate next generations. AI is often limited due to poor survival of cryopreserved avian sperm (Ushiyama et al., 2016). In this review, motility, viability and their fertilizing ability of sperm stored in vitro liquid conditions are reviewed with the knowledge of in vivo sperm survival mechanism(s). Moreover, other factors that may affect in vitro sperm motility, viability and fertilizing ability are also considered in this study. Probably, no study has been conducted earlier which considered most of these factors related with sperm motility, viability as well as fertilizing ability in vitro conditions. This study will help to understand an overall scenario of in vitro avian sperm motility, viability and their fertilizing ability.
2. Motility, viability, and fertilizing ability of avian sperm in different physiochemical conditions
2.1 Poultry semen extenders
Poultry semen extender is liquid in nature that protect the sperm and extends their longevity in vitro with a view to maintaining sperm fertilizing ability. The ingredients and composition of extenders varies from one extender to another. There are some well-known extenders that are using widely in poultry industry. Among the commercial extenders, Beltsville poultry semen extender, EK extender and Lake extenders are widely used in poultry industry (Sexton, 1977; Sexton and Fewlass, 1978; Sexton and Giesen, 1982; Giesen and Sexton, 1983a; Giesen and Sexton, 1983b; Sexton and Giesen, 1983; Morrell et al., 2005; Siudzińska and Łukaszewicz, 2008). Sperm viability and fertilizing ability varies from one extender to another extender. The breeders compared different poultry semen extenders with different poultry species to find out the suitable poultry semen extender (Sexton, 1988; Iaffaldano et al., 2016; Rakha et al., 2016). Still now, there is no reliable extender that can fully protect the sperm in vitro condition, as sperm quality deteriorate on storage time. For achievement of better fertility, two-stages extender also developed in chicken. The first stage reduces sperm metabolism and motility during storage time and second stage for achieving motility prior to insemination (Ameha et al., 2007). To maximize the keeping quality of sperm, the researchers are trying to develop most appropriate semen extender by applying the knowledges of in vivo sperm storage mechanism(s) and sperm physiology.
2.2 Dilution of sperm
The ejaculate volume and sperm concentration varies among avian species (García-Herreros, 2016). Generally, avian sperm are highly concentrated, low in volume and containing 6 (roosters) to 12 (toms) billion sperm/ml (Donoghue and Wishart, 2000). During in vitro storage condition, the percentage of viable sperm is lower in undiluted semen as compare to semen diluted with extender (Dumpala et al., 2006). So, the undiluted sperm needs to be diluted for in vitro storage or AI purpose. The sperm dilution rate, keeping quality and fertilizing ability varies among avian species. In broiler breeder, dilution of semen prior to storage is important for maintaining sperm quality index and semen diluted at 8 to10-folds maintained sperm fertilizing ability (Parker and McDaniel, 2004). It is also important to be noted that sperm fertilizing ability declines at higher dilution rate and, especially, fertility is drastically reduced at dilution rate of 1:10 or 1:20 (Wilcox, 1958). The guineafowl which is known for lower fertility rate as compare to other avian species, they can also maintain sperm survivability and fertilizing ability at low dilution rate (1:2) (Hudson et al., 2016). Probably, most detrimental effect of sperm dilution is known as dilution effect. It is possible that extensive dilution may destabilize sperm membrane which is detrimental for sperm motility and viability (Maxwell and Johnson, 1999). For achieving the maximum fertility in chicken, dilution effects was adjusted with sperm number (Sexton, 1981). So, the adjustment of sperm dilution is important in avian species for achieving maximum viability and fertilizing ability in vitro condition.
2.3 Effect of temperature
In avian species, sperm production, transportation and maintenance in the male reproductive tract occurs at body temperature 41°C (Beaupré et al., 1997). During mating, the sperm enters into SSTs of female reproductive tract, and the sperm can maintains its fertilizing ability at body temperature 41°C (Sasanami et al., 2013). The sperm of male reproductive tract (testis, epididymis and ductus deferens) are almost immotile at 40 °C as undiluted sperm, but restart motility when reduces the temperature to 30 °C or diluted with diluents (Ashizawa and Sano, 1990). Sperm motility, viability and fertilizing ability of different poultry species varies with different storage temperature. Sperm motility, viability and fertilizing ability pattern in different poultry species are also somehow different. Effect of temperature on motility, viability and fertilizing ability of avian sperm have been shown in Table 1. Briefly, in vitro avian sperm motility, viability and fertilizing ability is related with sperm storage temperature. Although sperm survives at body temperature 41 °C in both male and female reproductive tract, but this ability is lost within a few hours when sperm are incubated at 41 °C in vitro condition. Generally, sperm motility is low at both high and low temperature. The sperm motility is high at temperature between 20-37 °C. In case of sperm viability study, high percentage of viable sperm usually found at low temperature (approximately 4 °C). It is possible that metabolic activity of spermatozoa decreases at low temperature and this condition might be associated with long-term sperm viability in vitro condition. Sperm storage at 5 °C for 18 h provides similar fertility like fresh semen in turkey and decreased fertility at 15 °C, 25 °C and 35 °C (Giesen and Sexton 1983a).
2.4 pH of extender
Intracellular pH regulation is important for functioning of sperm (Nishigaki et al., 2014). In avian species, the optimum semen pH usually ranges between 7.0 and 7.4 (Siudzińska and Łukaszewicz, 2008; Ondho, 2014; Getachew, 2016). The pH in the lumen of Japanese quail SSTs is < 6.0, where sperm remains in a quiescence state of motility (Matsuzaki et al., 2015). During in vitro sperm storage condition, sperm motility and velocity decreases at lower pH and increases the sperm motility at higher pH in avian species. The effect of pH on motility, viability and fertilizing ability of avian sperm have been shown in Table 2. Motility, viability and fertilizing ability of avian sperm is affected by pH of extender. pH of semen is likely to be alkaline in state and the sperm transferred into SSTs where acidic state prevails. Sperm activity increases in alkaline pH, whereas activity decreases in acidic pH. At low pH, sperm flagellar quiescence happens with low sperm motility, and this condition might be associated with long-term sperm viability. Thus, a decrease in pH could play an important role in the process of long-term sperm viability in vitro condition.
Table 1: Effect of temperature on motility, viability and fertilizing ability of avian sperm
||Temperature dependent sperm functions
||sperm motility high at 20-37 °C
sperm motility almost inhibited at 5 °C and 41°C
|Ashizawa and Nishiyama, 1978
||high percentage of death sperm and low quality of sperm index at 41 °C as compare to 4 °C and 21 °C
||Dumpala et al., 2006
||sperm motility and fertility high at 5 °C and low at 41 °C
||Clarke et al., 1982
||sperm viability similar at 5 °C and 15 °C after 24 h storage
||low fertility at 25 °C as compare to 10 °C
||complete inhibition of sperm motility at 40 °C compare to 30 °C
||Wishart and Wilson, 1999
||sperm motility decreases at 40 °C compare to 30 °C
||Wishart and Wilson, 1999
||Sperm quality high at 15 °C as compare to 10 °C
||Carter et al., 1957
||sperm motility and fertility low at 41 °C
sperm motility high at 5 °C but fertility high at 25 °C
|Clarke et al., 1982
||sperm motility almost similar at 30 °C and 40 °C
||Wishart and Wilson, 1999
||sperm motility increases at 40 °C compare to 20 °C
||Bonato et al., 2012
2.5 Effect of osmolarity
Table 2: Effect of pH on motility, viability and fertilizing ability of avian sperm
|5.8, 6.8, 7.1 and 7.4
||sperm storage at 5 °C for 24 h
||most satisfactory fertility was found at pH 6.8 or 7.1
||Lake and Ravie, 1976
|6.0, 7.0 and 8.0
||sperm storage at 7-9 °C for 48 h
||motility highly preserved at pH 6.0
||Bogdonoff and Shaffner, 1954
|5.0, 6.0, 7.0, 8.0 and 9.0
||Chicken, Turkey and Quail
||sperm storage at 30°C for 3 h
||sperm were immotile up to pH 6 and motility increases with pH
||Holm and Wishart, 1998
|5.5, 6.0 and 6.5
||sperm storage at 15 °C for 6 h
||motility and fertilizing capacity decreases at pH 5.5 compare to pH 6.0 and 6.5
||Sexton and Giesen, 1983
|6.0, 7.0, 8.0 and 9.0
||sperm storage at 20 °C and 40 °C for 10 min
||sperm viability decreases with the increase of pH
||Bonato et al., 2012
Osmotic condition is associated with the intactness of sperm plasma membrane. In avian species, sperm viability was studied with wide range of osmolarity at different storage conditions. It reveals that very high and low osmolarity compare to isotonic conditions hampers sperm survivability. In chicken and turkey, sperm viability adversely affected when osmolarity was ≥ 800 mOsm/kg. Even after short storage period of 10 min, sperm survivability was significantly low in chicken and turkey at 50 mOsm/kg and ≥ 800 mOsm/kg (Blanco et al., 2000). In hypo-osmotic condition, sperm below 200 and 140 mOsm/kg adversely affected the fertility of chicken and turkey spermatozoa, respectively (Bakst, 1980). Turkey sperm osmotolerance was slightly improved by reducing the incubation temperature from 21 °C to 4 °C (Blanco et al., 2008). In emu species, spermatozoa can tolerate to osmolarities as high as ≈ 1400 mOsm/kg but lost the motility score after > 700 mOsm/kg (Sood et al., 2011). Fertility and hatchability of chicken sperm stored under hypertonic (460 mOsm/kg) or isotonic (340 mOsm/kg) condition did not differed after 24 h of sperm storage (Van Wambeke, 1977). The osmolarity of avian seminal plasma is like to be isotonic (330.4±50.4) (Siudzińska and Łukaszewicz, 2008; Dietrich et al., 2010). Conclusively, avian sperm may survive in a range of osmolarity between 250 and 450 mOsm/kg.
3. Addition of additives to sperm storage conditions
3.1 Role of seminal plasma
Seminal plasma (SP) is a complex fluid (Poiani, 2006), varies among species and plays an essential role for sperm functioning in male and female reproductive tract (Juyena and Stelletta, 2012). Most components of SP are secreted from primary sex glands (seminal vesicle, prostate gland and bulbourethral gland) in mammals (Mann and Lutwak-Mann, 1951; Maňásková et al., 2002; Duncan and Thompson, 2007) and insects (Happ, 1984; Gillott, 1996). Unlike mammals and insects, avian species lack the prostate gland, seminal vesicle and bulbourethral gland. Some avian species produces lymph-like fluid and/or foam at the time of ejaculation (Fujihara, 1992). Male quail produces large quantities of cloacal foam at the time of ejaculation (Seiwert and Adkins-Regan, 1998). Thus, in avian species, the fluid adds with the sperm mainly comes from the male reproductive tract and cloacal region. As an avian species, the sperm production, processing and transportation in male Japanese quail have been shown in Fig. 1.
The roles of SP in in vitro sperm survivability and fertility are well studied in mammalian species (Thaler, 1989; Killian et al., 1993; Manjunath and Thérien, 2002; Moura et al., 2006; Manjunath et al., 2007; Koppers et al., 2011; Juyena and Stelletta, 2012; Crawford et al., 2015; Viana et al., 2018) and insects (Bertram et al., 1996; Lung et al., 2001; Bloch Qazi and Wolfner, 2003; Holman, 2009). The studies related to in vitro sperm survivability and fertilizing ability in avian species are limited and are shown in Table 3. Probably, the first proteomic analysis of SP was investigated in rooster semen (Marzoni et al., 2013). The role of SP in avian sperm viability and fertilizing ability study seems to be contradictory. Blesbois and de Reviers, (1992) reported that fowl SP contains the fraction of higher molecular weight (>50 kDa) favours sperm fertilizing ability whereas fractions of lower molecular weight (<1 kDa) are toxic to sperm in vitro condition. In turkey, whole SP reduces fertility (Douard et al., 2005), whereas dialyzed SP at 12-14 kDa is beneficial for in vitro sperm viability (Iaffaldano and Meluzzi, 2003). Sexton, (1977) reported that removal of seminal plasma by centrifugation had no significant effect on fertilizing capacity of chicken sperm at 5 °C for 24 h. It is possible that SP contains several components and all components are not beneficial for sperm viability and fertilizing ability. Proteomic analysis of SP and the application of SP components in sperm viability test may give clearer scenario about the role of SP in avian species.
Figure1: Sperm production and transportation process in male Japanese quail. The figure is prepared from the findings of Clulow and Jones (1982). Sperm produces in testis, matures in epididymis and briefly store in ductus deferens. Sperm production, maturation and transportation is relatively quicker in Japanese quail. During ejaculation, cloacal foam is added with the semen. Testicular and epididymal sperm show low motility compare to sperm of ductus deferens and ejaculates.
3.2 Role of antioxidants
Table 3: Effect of seminal plasma on motility, viability and fertilizing ability of avian sperm
|Type of SP
|Fraction of SP (Mr <1000)
||washed spermatozoa at 41 °C for 15 min
||stimulate sperm motility and oxygen consumption
||Ashizawa and Okauchi, 1984
|Fraction of SP (Mr <1 kDa and >50 kDa)
||sperm stored at 4 °C for 24 h
||<1 kDa decreases fertility
>50 kDa increases fertility
|Blesbois and de Reviers, 1992
|SP + PBS (1:2)
||sperm stored at 0 °C for 24 h
||inhibit endogenous lipid peroxidation in sperm and improve fertility
||Fujihara and Koga, 1984
|SP albumin (1 or 4 mg/ml)
||sperm stored at 4 °C for 24 h
||sperm mobility stimulating factor
||Blesbois and Caffin, 1992
||sperm stored at 4 °C for 24 h
||Douard et al., 2005
|SP dialyzed at 12-14 kDa
||sperm stored at 5 °C for 24-48 h
||improve sperm viability, membrane integrity and sperm motility
||Iaffaldano and Meluzzi, 2003
Naturally, avian semen contain antioxidants and enzymatic defenses that prolongs the sperm longevity in vivo and in vitro condition (Bréque et al., 2003; Partyka et al., 2012). The amount of antioxidants and antioxidant enzyme present in avian seminal plasma and sperm have been shown in Table 4. Avian sperm also containing beta-defensins, an antimicrobial peptide which is probably associated with the protection of sperm from microbial infection in the male and even in female reproductive tract (Shimizu et al., 2008; Das et al., 2011). However, avian sperm contains high amount of polyunsaturated fatty acids which is susceptible to reactive oxygen species and promotes lipid peroxidation (Khan, 2011). During in vitro storage condition, total lipid contents of spermatozoa decrease significantly at 2-5 °C after 48 h in chicken. It is possible to occur lysis of lipid and peroxidation; and can modify the structure of spermatozoa (Blesbois et al., 1999). In turkey, phospholipid content of spermatozoa decreases after 48 h sperm storage at 4 °C (Douard et al., 2000). To overcome the problem of lipid peroxidation and achieving better fertility, experts tried to add different antioxidants as feed additives (Rengaraj and Hong, 2015; Tufarelli and Laudadio, 2016; Surai et al., 2019). Here, effect of different antioxidants on in vitro sperm storage are discussed only. Poultry breeders are using different kinds of antioxidants in vitro sperm storage condition to improve the sperm quality. The effect of antioxidants on motility, viability and fertilizing ability of avian sperm have been shown in Table 5. It can be said that antioxidant improves motility, viability and fertilizing ability of avian sperm in a dose dependent manner.
3.3 Effect of serum
Table 4: Antioxidant and antioxidant enzymes in avian seminal plasma and sperm
|Total antioxidant activity in seminal plasma
|0.62 ± 0.02a
||2.10 ± 0.14a
||13.15 ± 1.11a
||2.13 ± 0.11a
||0.97 ± 0.04a
||0.8 ± 0.3b
|Superoxide dismutase activity in seminal plasma (U/ml)
||46.20 ± 2.44a
||32.27 ± 3.11a
||77.71 ± .07a
||42.05 ± 3.06a
||65.98 ± 1.54a
|Superoxide dismutase activity in sperm
|Glutathione peroxidase activity in spermatozoa (U/109 sperm)
|Glutathione peroxidase activity in seminal plasma (mU/ml)
||12.7 ± 3.0c
||28.7 ± 7.8c
|Vitamin E in seminal plasma (ng/ml)
||163.7 ± 6.5d
||32.4 ± 2.6d
|Vitamin E in sperm
|182.48 ± 8.5d
||48.1 ± 2.4d
Serum, especially bovine serum albumin (BSA) has been used widely in mammalian sperm viability study in vitro condition. BSA plays important role in sperm capacitation (Xia and Ren, 2009) and acrosome reaction (Hossain et al., 2007) in mammals. BSA improves motility and viability of frozen-thawed ram spermatozoa (Uysal and Bucak, 2007), extend sperm longevity and maintains sperm plasma membrane integrity (Tvrda et al., 2010; Sarıözkan et al., 2013) and can be substituted for egg-yolk in ram semen diluents (Matsuoka et al., 2006). The studies related to effect of serum in sperm viability and fertilizing ability is limited in avian species. In turkey, addition of BSA with diluents significantly improve the sperm motility and velocity at 7 °C after 24 h of sperm storage (Bakst and Cecil, 1992). BSA increases the viability of rooster sperm in vitro condition (Kim et al., 2017).
3.4 Effect of cloacal gland secretions
Table 5: Effect of antioxidants on motility, viability and fertilizing ability of avian sperm
||sperm storage at 4 °C for 24 h
||improves sperm progressive motility, viability and reduces morphological defects
||Tabatabaei et al., 2011
||sperm storage at 4 °C for 72 h
||improves sperm motility and viability
||Asmarawati and Yuwanta, 2010
||vitamin E 8 µg/ml, sperm storage at 4 °C for 24 h
||improves fertilizing ability
||Blesbois et al., 1993
||sperm storage at 4 °C for 24 h
||no reduction of lipid peroxidation
||Long and Kramer, 2003
|Vitamin E, BHT and TEMPO
||sperm storage at 5 °C for 48 h
||improves sperm viability and plasma membrane integrity
||Donoghue and Donoghue, 1997
||lycopene 500 µg/ml, sperm storage for 24 h
||improves sperm viability
||Mangiagalli et al., 2007
Cloacal foam is secreted from the cloacal gland of adult male Japanese quail and introduces into the female reproductive tract during natural mating. It was reported that removal of cloacal gland secretions from the male before mating reduced the fertility, but the supplementation of cloacal gland secretion rescued the subfertility (Sasanami et al., 2015). In vitro sperm motility study was conducted with the cloacal gland secretion and found that 5% cloacal gland secretion enhanced quail sperm motility for short time of 2-3 h at room temperature (Biswas et al., 2010). Interestingly, the sperm motility was inhibited at high concentration (25%) of cloacal gland secretions (Biswas et al., 2010). It is reported that cloacal gland secretion promotes sperm motility and act as a medium to facilities sperm transport in the oviduct (Singh et al., 2011; Cheng et al., 1989).
3.5 Effect of utero-vaginal junction extract
Avian sperm can be stored in the specialized structure of SSTs, which is situated inside the utero-vaginal junction (UVJ) of oviduct (Bakst, 2011) and may protect themselves from adverse conditions in SSTs (Das et al., 2006). It is known that sperm remains in the SSTs as a quiescence state in motility (Matsuzaki et al., 2015). The molecular mechanism behind the long-term sperm storage in the SSTs and retention of fertilizing ability after a single insemination still mystery. It is believed that SSTs contains some molecules and have some mechanism(s) that facilitate sperm storage and fertilizing ability in vivo condition. In this regard, in vitro sperm motility and viability was conducted with the fluid collected from reproductive tract. Fluid from ovarian pocket of hen stimulate sperm motility at body temperature and the fluid may facilitate the fertilization process (Ashizawa and Wishart, 1992). Albumin and transferrin were isolated from UVJ of quail and their effects on in vitro sperm survivability was significantly high when compared with control (Matsuzaki et al., 2019).
Conclusively, it can be said that sperm motility, viability and fertilizing ability in avian species depends on some physiochemical properties. Generally, combination of low pH and low temperature may reduce motility and metabolic activity of sperm. The addition of additives may improve the sperm quality in a dose dependent manner. Moreover, further studies are needed to find out the mechanism(s) behind sperm storage in vivo condition. Application of research findings into in vitro storage conditions may enrich sperm motility, viability and fertilizing ability in avian species.
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