Abstract: This study was conducted to identify the suitable diluent for short-term preservation (0, 24 and 48 h) of Guinea fowl (pearl variety) semen. Semen samples were collected and diluted with normal saline, CARI, BPSE and Lake’s semen diluents and kept at 6°C for storage. No significant difference was found in sperm motility among the different diluents at 0 h (freshly ejaculated) storage period. A significant (p<0.05) reduction was found in sperm motility among all the diluents at 24 h storage period. Subsequently, there was no sperm motility was recorded in normal saline diluted semen samples at 48 h storage period. While a reduction (p<0.05) in sperm motility was recorded in rest of the three semen diluents. The mean values for fertility with freshly ejaculated semen during 2-12 days of fertile period were recorded as 66.27±4.08, 78.90±3.08, 64.90±4.87 and 67.99±6.36% whereas, after 24 h storage of semen, fertility reduced drastically such as: 0, 41.97±7.27, 36.09±8.72 and 30.90±7.12% in normal saline, CARI, BPSE and Lake’s semen diluent, respectively. No fertility was noticed at 48 h storage in any semen diluent. Irrespective of the storage period and diluents, CARI semen diluent expressed better fertility than others in Guinea fowl.
INTRODUCTION
In some parts of the world Guinea fowls are an important source of high quality protein in terms of eggs and meat but the supply of these products are not regular because the birds are seasonal breeders. In addition, the behaviour of male Guinea fowl is monogamous which drastically limits the reproductive efficiency of the bird especially when the male bird dies or culled. Under these conditions female is not readily copulate naturally with a new male (Aire et al., 1983; Biswas, 1983). Hence, to supply the high quality protein to the growing human population and to fight against the malnutrition, reproductive efficiency of guinea fowl must be improved by breaking the seasonality and adopting the Artificial Insemination (AI) in this bird.
Ivanov (1907) reported first AI in bird. Since then, AI has gained momentum as a valuable breeding tool. Superiority of AI to natural mating was noticed by Saeki and Nagomi (1964) from analysis of four years of comparative data collected from domestic fowl in Japan. Further success of this technique needs suitable semen diluent. Diluents are essential for semen in order to increase number of birds inseminated by per unit volume of semen and it prolongs the sperm survival under both short and long term preservation of semen in vitro. Considerable information is available on the semen diluent of chicken and turkey (Lake, 1960; Van Wambeke, 1967; Sexton, 1977; Lake and Ravie, 1981; Mohan et al., 2011) and frequently used wherever the technique of AI is employed. Studies indicated that most of the semen diluents were targeted for chicken and turkey and there is no proper diluent for guinea fowl semen. Hence, the information is scanty for the specific diluent of Guinea fowl semen. As to improve the reproductive efficiency of the bird, this study was carried out to select the suitable diluent for Guinea fowl from available poultry semen diluents such as: CARI semen diluent (Mohan et al., 2000), Beltsville Poultry Semen Extender (BPSE) (Sexton, 1977) and Lake’s semen diluents (Lake, 1960). Normal saline was also taken as a diluent in this investigation because it is routinely employed for diluting the avian semen (Shinde et al., 2013; Ogbu et al., 2014).
MATERIALS AND METHODS
Sixty healthy and adult female Guinea fowl (pearl variety) from the same hatch were taken randomly and maintained in individual cages under uniform husbandry conditions. For the fertility study, thirty healthy and adult males from the same hatch of the same variety were taken and maintained in the similar manner. They were given normal breeder ration and water ad libitum with a constant light of 14 h day–1. Semen samples were collected every alternate day during study period by abdominal massage method (Burrows and Quinn, 1935). Pooled semen of Guinea fowl was diluted in various semen dilutor such as normal saline (0.89% (w/v) NaCl), CARI diluent (Mohan et al., 2000) with modifications (under process of patent), Beltsville Poultry Semen Extender (BPSE) (Sexton, 1977) and Lake’s diluent (Lake, 1960). One part (1 mL) of good quality semen was taken in 5 mL round bottom glass tube (length = 7 cm, diameter = 1 cm) and mixed with equal volume of the respective diluent. Artificial insemination was carried out in birds at 3 different time intervals (0, 24 and 48 h) after semen collection. Diluted semen samples targeted for AI were stored at 6°C for 24 and 48 h.
Sixty hens of guinea fowl were equally divided in to 4 groups with 15 birds in each. Group 1, 2, 3 and 4 of hens were inseminated with the semen diluted (1:2) with normal saline, CARI, BPSE and Lake’s diluent respectively. Before AI diluted semen of each group was examined for sperm motility at different storage period (0, 24 and 48 h). It was determined under low magnification (10X) of light microscope with cover slip and scored as described by Wheeler and Andrews (1943). For artificial insemination, vaginal eversion was made by gentle pressure on left side of the abdomen through the cloaca (Quinn and Burrows, 1936). Subsequently, with the help of tuberculin syringe which contain the doses (60-100 million spermatozoa) of good quality diluted semen, insemination was done (0.1 mL) into the well everted oviduct (to the depth 3-4 cm or as close as possible to the sperm host gland) and concurrently with the release of pressure on the abdomen so that oviduct revert to its normal position.
Fertility of birds was assessed by incubating the eggs (99.5°F temperature and 55-60% relative humidity) laid by hens from 2-12 days after single intra vaginal insemination. To determine the fertility, eggs were examined by candling at 9th day of incubation. Some eggs were broken for fertility assessment in which fertility was not clear by candling. The percent fertility was calculated as a ratio of number of fertile eggs to the number of total egg set in the incubator. Statistical analysis was done using statistical software package (SPSS-16) for ANOVA (Snedecor and Cochran, 1994) and Duncan’s multiple range tests (Duncan, 1955) by comparing means for significant differences.
RESULTS AND DISCUSSION
The mean values of sperm motility in guinea fowl are presented in Table 1. All the semen diluents expressed the sperm motility between the ranges of 82.00±1.79-87.46±2.80% at 0 h of storage. There was no significant difference among the different diluents at this storage period. Shinde et al. (2013) conducted the study on chicken semen storage using various diluents and found no significant difference in sperm motility among the different diluents. At 24 h of storage, a significant (p<0.05) reduction was found in sperm motility among all the diluents. Similar results were observed by Mohan et al. (2013) using CARI diluent after 24 h storage of guinea fowl semen. Subsequently, no sperm motility was recorded in normal saline diluted group at 48 h of storage at low temperature. This may be due to the fact that normal saline is lack of energy source (sugar) and constituents like glutamic acid responsible for maintaining osmotic pressure of avian semen (Lake and McIndoe, 1959; Sexton and Fewlass, 1978). A significant reduction in sperm motility during 24/48 h storage was also recorded in rest of the three semen diluents (Table 1). Similar results were obtained in chicken by other workers (Shinde et al., 2013). 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). Therefore, to store Guinea fowl semen, the type of diluent and storage temperature is very important to avoid a reduction in sperm quality.
Irrespective of the diluents, sperm motility (Table 1) and its fertilizing ability (Table 2 and 3) showed a declining trend as storage time progresses from 0-48 h. The mean values for fertility with freshly ejaculated semen during 2-12 days of fertile period were recorded as 66.27±4.08, 78.90±3.08, 64.90±4.87 and 67.99±6.36% by employing normal saline, CARI, BPSE and Lake’s semen diluent respectively (Table 2). During the same fertile period after 24 h storage of semen, fertility was reduced markedly such as: 0, 41.97±7.27, 36.09±8.72 and 30.90±7.12% in normal saline, CARI, BPSE and Lake’s semen diluent, respectively (Table 3). This suggested that fertilizing ability of Guinea fowl spermatozoa reduced drastically during low temperature storage. Overall, during short term storage (24 h) of Guinea fowl semen CARI diluents exhibited superior pattern of fertility (Fig. 1) than others (Table 2 and 3).
| Table 1: | Effect of different semen diluents on sperm motility of Guinea fowl spermatozoa |
| BPSE: Beltsville poultry semen extender, Means bearing different superscript (a,b,c) within columns differ significantly (p<0.05), Means bearing different superscript (1,2) within rows differ significantly (p<0.05), (Mean±SEM, n = 6) | |
| Table 2: | Effect of different semen diluents on fertilizing ability of 0 h stored Guinea fowl spermatozoa |
| BPSE: Beltsville poultry semen extender, Means bearing different superscript (a,b) within columns differ significantly (p<0.05), Means bearing different superscript (1,2) within rows differ significantly (p<0.05), (Mean±SEM, n = 5) | |
| Table 3: | Effect of different semen diluents on fertilizing ability of 24 h stored Guinea fowl spermatozoa |
| BPSE: Beltsville poultry semen extender, Mean values bearing different superscripts (a,b) in column differ significantly (p<0.05), (Mean±SEM, n = 5) | |
| Fig. 1: | Fertilizing ability of Guinea fowl spermatozoa after single insemination using CARI semen diluent (Mean±SEM) |
These results are in agreement with the work of Shinde et al. (2013) carried out in chicken using various semen diluents. Kammeree et al. (1972) found that the number of progressively motile sperm per ejaculate was the most consistent and reliable trait correlated with fertility. Positive correlations between motility and fertility have been reported by several investigators (Kamar, 1960; McDaniel and Craig, 1962; Kammeree et al., 1972; Monsi et al., 1975). Hence, reduction of motility (Table 1) with the advancement of storage period of the spermatozoa either in vivo (sperm storage tubules) or in vitro (at 6°C) may be the reason for decline in fertility (Table 2 and 3) in Guinea fowl. Absolutely no fertility was noticed when 24 h stored normal saline diluted semen was inseminated to the hens. As discussed earlier that normal saline is fluid which is deficient of energy and buffering capacity. Further, it is unable to maintain osmotic pressure of spermatozoa during short term storage at low temperature.
From the present investigation, it is concluded that to store Guinea fowl semen, the type of diluent and storage temperature plays a crucial role in storage of Guinea fowl semen as indicated by the sperm quality. Further, it is interpreted that at all the storage period (0 and 24 h), CARI semen diluents expressed higher fertility than others. This suggests that the ingredients of CARI diluent are comparatively more suitable to the survival of spermatozoa of Guinea fowl.