Efficiency of Different Diluents and Dilution Rates on the Fertilization Potential of Chicken Spermatozoa

Artificial Insemination (AI) is an incredible tool that revolutionized poultry breeding. Extensive use of AI led to various studies on seminal diluents driven by low volume, substantially viscous and concentrated chicken semen. In general, in vitro semen preservation is intended to mimic the biological environment of the hen’s reproductive tract where the survival of spermatozoa is prolonged through certain mechanisms like reversible suppression of sperm respiration and motility; stabilization of plasmalemma and enzyme systems and suppression of sperm immunogenicity (Bakst 1993). The diluents are commonly based on the biochemical constituents of seminal plasma which includes energy sources, buffered salts and osmotic regulators. Glucose and/or fructose are the common energy providers used in semen extenders. While salts of phosphates, carbonates and zwitterions like BES [N,N-bis (2-hydroxyethyl) -2-aminoethane-sulfonic acid] or TES [N-tris (hydroxymethyl) methyl-2-aminoethane-sulfonic acid] contribute to buffer action, salts of glutamate, acetate, citrate and chloride maintain the osmotic pressure of  extenders (Donoghue and Wishart, 2000). Various diluents were explored till date from a simple saline or ringer’s solution to a much complex composition. Nevertheless the many diluents commonly formulated to hold semen for a few hours to days between 0 and 5°C, oftentimes resulted in diminishing fertility with time in a species specific manner. This has not changed the insight of liquid semen storage in poultry because of the physiological uniqueness of cockerel sperm having less cytoplasmic volume and longer tail length, limiting its vitality in post freeze/thawing as in cryopreservation (Thurston and Hess, 1987; Kuzlu and Taskin, 2017). Apart from minimizing the sperm dose per insemination, the effective use of extenders and optimal dilution rate will reduce the deleterious factors present in the seminal plasma and complement the natural protective elements of the ejaculate.
       
Every semen extender is reported to be discrete in maintaining sperm integrity, causing wide range of variations in fertility. The ICAR-CARI (Central Avian Research Institute, Izatnagar, U.P., India), has also developed a simple diluent requiring adjustments neither in pH nor in osmolarity, named ‘CARI poultry semen diluent’ (Mohan et al., 2017). Having been compared with the chicken diluents like BPSE (Beltsville Poultry Semen Extender) and Lake’s in the earlier works (Shinde et al., 2013; Mohan et al., 2015), the present work is designed to study different semen extenders such as EK and Tselutin extender with CARI poultry semen diluent during short term preservation and to ascertain the influence of different dilution rates on the fertility of White Leghorn chicken spermatozoa.

Experimental design and husbandry practices
 
The proposed experiment was conducted in the Division of Avian Physiology and Reproduction, Central Avian Research Institute, Izatnagar, Bareilly, U.P. The institute is situated at an altitude of 169 m above the mean sea level, at latitude of 28°N and longitude of 79°E. The place experiences extreme hot (45°C approx) and cold (5°C approx) conditions with the relative humidity ranging between 15 to 85%.
       
Twenty healthy and sexually mature breeding cockerels pertaining to the same hatch of White Leghorn (WLH) chicken were reared in individual cages under uniform management conditions, as per the guidelines and approval of Institute Animal Ethics Committee. To assess fertilizing ability, 60 healthy sexually mature WLH females divided into 3 groups with 20 birds each from the same hatch were chosen and raised under similar conditions. All experimental birds were offered normal breeder ration, ad libitum water and maintained in a regime of 14 h constant light every day.
 
Collection of semen
 
The cockerels were familiarized with semen collection on every alternate day through abdominal massage technique (Burrows and Quinn, 1937). During the study period precautions were taken to avoid contamination of semen with fecal matter, urates and transparent fluid which deteriorate the semen quality.
 
Short term preservation using different diluents
 
Pooled semen samples were divided equally into three aliquots and were taken separately in 5ml round bottom glass tubes (length 7cm, diameter 1cm). Each fraction was diluted with three different semen extenders - CARI poultry semen diluent containing Dipotassium hydrogen phosphate, Potassium hydrogen phosphate, D-Fructose, Sodium glutamate, Sodium acetate, N-Tris, Tri potassium citrate, Magnesium chloride and Sodium bicarbonate of pH 7.2 and Osmolarity 320 mOsmol/kg (Mohan et al., 2017); EK extender (g/dl) containing Potassium citrate 0.14, Sodium dihydrogen phosphate 0.21, Disodium hydrogen phosphate 0.98, Glucose 0.70, Sodium glutamate 1.40, D-fructose 0.20, Inositol 0.70, Polyvinylpyrrolidone 0.10 and Protamine sulphate 0.020 of pH 7.5 and Osmolarity 390 mOsmol/kg (Lukaszewicz, 2002); Tselutin extender (g/dl) containing D-fructose 0.80, Protamine sulphate 0.032, Sodium glutamate 1.92, Potassium acetate 0.50 and Polyvinylpyrrolidone 0.30 of pH 7.1 and Osmolarity 320 mOsmol/kg (Tselutin et al., 1995) at a fixed dilution rate (1:2) for AI at 0 h and 24 h. Diluted semen samples targeted for AI at 24 h were stored at 8±1°C for CARI poultry semen diluent and 4±1°C for EK and Tselutin extenders. The chemicals used in diluent preparation were of analytic grade and supplied by Sigma-Aldrich.
 
Motility and fertility assessment
 
Before AI, the diluted semen samples were examined for sperm motility at different storage periods viz: 0, 24, 48, 72 and 96 h. It was tested under light microsope (10x) using cover slip and graded as stated by Wheeler and Andrews (1943). Thereafter AI was carried out by intravaginal insemination (80 to 100 sperm/hen) through vaginal eversion (Burrows and Quinn, 1936) using AI gun (IMV, France) in all the 3 groups with the respective diluted semen samples. The day next to AI was skipped and eggs were collected from 2 to 8 days after insemination. Fertility of eggs was examined by candling on 9^th day of incubation. Breakout analysis was performed in few eggs in which fertility was doubtful upon candling. The percent fertility was measured by dividing the total number of fertile eggs by the number of total eggs set in the incubator, multiplied by 100.
 
Semen dilution at different dilution rates
 
When the research was further extended to analyse the influence of different dilution rates on fertility using fresh semen, 20 healthy and sexually mature white leghorn cockerels and 200 healthy adult females divided into 10 groups with 20 birds each were selected. Other criteria were similar to the experimental birds mentioned above. Semen samples were pooled and diluted with CARI poultry semen diluent at different dilution rates of 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18 and 1:20 with gradual decrease in the sperm concentration as shown in Table 1. Motility assessment of sperm was determined at 0, 24 48, 72 and 96 h of storage periods (8±1°C) under low magnification as described by Wheeler and Andrews (1943). The sperm concentration (million per ml) was determined from the standard calibration curve established between absorbance and sperm concentration in a double beam UV-VIS spectrophotometer at 550 nm (Brillard and McDaniel, 1985). The calculated doses of samples were appropriately taken in separate 5 ml round bottom glass tubes for AI at 0 h (freshly ejaculated semen). Intra vaginal insemination was carried out in the respective group of hens with AI gun (IMV, France) with 50 µl volume of inseminate using various dilution factors. The day next to AI was skipped and eggs were collected from 2 to 8 days post insemination. Fertility assessment was done same as described above.
 

 
Statistical analysis
 
The data were subjected to analysis of variance using statistical software package SPSS (version 20) and the significant differences among means were determined by Duncan’s multiple range tests (Duncan, 1955).

The mean values of sperm motility (%) recorded in WLH chicken spermatozoa are presented in Table 2. At 0 h of storage, the undiluted semen and all the diluent groups expressed the sperm motility between 89.33±0.49 to 90.33±0.67, showing no significant difference. Previous studies also found insignificant difference in the initial sperm motility of Indian reared white leghorn and the native chicken (Mukherjee and Bhattcharya, 1949; Nayak et al., 1990; Shinde et al., 2012). At 24 h of storage, a significant reduction (p<0.05) in sperm motility was found among all the groups except CARI semen diluent with the lowest in undiluted semen (10.17±0.65%). Further, a drastic reduction in sperm motility was observed with time (48 to 96 h) in all the diluent groups, with undiluted semen showing nil motility. It is known that sperm motility and the fertilizing ability of undiluted neat fowl semen stored in vitro usually decreases within 1 h of collection (Carter et al., 1957). Any decrease in semen quality upon storage in reduced temperature is ascribed to the apoptopic changes and oxidative stress causing alterations in plasma membrane permeability and mitochondrial membrane potential (Vasicek et al., 2015). Here comes the decisive part of the extenders and storage temperature to sustain the seminal attributes against the time bound effects. The advantage of improved motility of diluent groups over the undiluted semen is because of the energy suppliers in the diluents in terms of glucose or fructose and moreover dilution hinders agglutination of sperms by reducing their concentration. Overall CARI poultry semen diluent expressed higher motility compared to other diluents.
 

       
In accordance with our findings, several researchers stated sperm motility as a credible trait correlated with fertility (McDaniel and Craig, 1962; Kammerer et al., 1972; Monsi et al., 1975). In comparison to the total fertile period (2 to 8 days), at 0 h of semen storage, higher fertility (%) was obtained during 2 to 6 days after AI from CARI poultry semen diluent (93±11.1) and EK extender (90.55±1.04) followed by Tselutin extender (Table 3). Upon AI of 24 h stored diluted semen, superior fertility was shown by CARI poultry semen diluent (90.10±1.55; 85.11±1.78) followed by EK (83.20±2.18; 77.68±1.79) over tselutin extender (76.13±1.41; 70.12±1.15) in 2 to 6 and 2 to 8 days of fertile periods respectively (Table 4). The key challenge of any semen extender is to retain the potency of spermatozoa in vitro in comparison to the storage in the oviduct system in vivo. Short term preservation (2 to 8°C) of spermatozoa (Donoghue and Wishart, 2000) in any extender containing substrates of glycolysis and tricarboxylic acid cycle, together support their long term viability in vitro. Similarly, the medium composition of CARI diluent with mixtures of acetate, citrate and glutamate along with phosphate and carbonate buffer action could be the reason for its supremacy over other diluents in the present study. Our findings were in line with the works of Shinde et al., (2013) and Mohan et al., (2015) which showed the superiority of CARI diluent over BPSE and Lake’s diluent in chicken and guinea fowl species respectively. Subsequently, the superiority of EK over Tselutin extender in our study is in agreement with Siudzinska and Lukaszewicz (2008). Though the ingredients of Tselutin extender overlap with EK, presence of both the energy sources (glucose and fructose) along with osmolyte inositol could be the reason for higher fertility of EK extender over Tselutin extender.
 

 

       
The impact of CARI diluent on per cent sperm motility displayed significant reduction with the increase in dilution and length of time from 24 to 96 h whereas no significance was noticed at 0 h (Table 5). The capability of a diluent to maintain the motility of sperms relies on multiple factors including semen viscosity, oxygen concentration, adenosine triphosphate (ATP) and ions like calcium and hydrogen (Parker and Mc Daniel, 2006). The fertility data (%) on the effect of different dilutions (1:2 to1:20) of fresh semen is presented in Table 6. The optimal fertility in our study at 2 to 6 days fertile period after AI was evidenced in 1:2 and 1:4 dilutions followed by 1:6 and 1:8 (p<0.05) with the record of 90% and higher fertility using the insemination dose as stated in Table 1. The dilution rates of 1:10 to 1:18 expressed fertility in the range of 79.13±1.83 to 87.23±1.35%. In the same fertile period, the study revealed about 77.33±0.49% fertility at the maximum dilution of 1:20 (12 million sperm/hen). A similar pattern of fertility was obtained from 2 to 8 days fertile period (Table 5). A recent work by Mohan and Sharma (2017) evinced higher fertility using CARI diluent (1:6) containing 38 million sperm per insemination dose. Thus a single cockerel ejaculate comprising 5.34 x 10⁹ sperm/ml, could cover 280 hens if collected on alternate days in a week, against the male to female ratio of 1:8 in natural mating, playing an immense role in economy. The reduction in sperm concentration alongside the elevated sperm respiration, higher metabolic activity and morphological changes are the potential explanation for the decline in fertility as with higher dilution rates in our research findings (Clarke et al., 1982). Additional cause of adverse effects at larger dilutions could be ascribed to the evasion of critical intracellular elements into the storage medium resulting from damage to the structural membrane lipids, or by the thinning of certain defensive and nutritional components in the seminal plasma (Blesbois and Brillard, 2007). Several investigators have attempted to determine the minimum insemination dose using a variety of extenders ranging from 24 to 200 million cells (Rowell and Cooper, 1960; Kim et al., 1974; Bandyopadhyay et al., 2006). Etches (1996) suggested 100 million cells per insemination for high fertility which is presently in use at the dilution rate of 1:2. These figures are also controlled by factors like type of diluent, dilution rate and storage conditions.
 

 

Under short term preservation, CARI poultry semen diluent exhibited higher fertility than other diluents considered for study and demonstrated superior fertility of 90% and higher upto 1:8 (29.70 million sperm/hen) dilution rate with fresh semen. The current research gives scope for obtaining optimum fertility levels in chicken at higher dilution rates than the conventional methods. As every extender has a distinct potential in preserving the seminal traits, species and breed specific investigations should be explored for successful storage and the advanced facilitation of assisted breeding techniques.

The authors are thankful to the Directors of ICAR- Indian Veterinary Research Institute and ICAR- Central Avian Research Institute for providing necessary research facilities.