EFFECT OF BEE HONEY AND/OR ROYAL JELLY ON THE FERTILIZING CAPACITY OF DILUTED RABBIT SEMEN STORED AT 5ºC

EFFECT OF BEE HONEY AND/OR ROYAL JELLY ON THE FERTILIZING CAPACITY OF DILUTED RABBIT SEMEN STORED AT 5ºC

 

A.M. El-Sherbiny

Animal Prod. Department, Faculty Agriculture, Ain Shams University, Cairo, Egypt.

 

The effect of bee honey and/or royal jelly (RJ) addition to tris-based extender on the motility, viability, abnormalities and fertilizing capacity of New Zealand White (NZW) rabbit spermatozoa was studied. Pooled semen was processed in tris-based extenders containing ascending concentrations (v/v) of bee honey (0, 1, 2, 3, 4, 5, 7.5 and 10% of diluent) and descending concentrations (v/v) of egg yolk (20. 19, 18, 17, 16, 15, 12.5 and 10%). RJ was added to tris-buffer extender with 2% bee honey and 18% egg yolk (EY), the concentrations (w/v) of RJ were 10, 20, 30, 40, 50 and 100 mg/6 mL diluent. Dilution rate was 1: 6 (1 semen + 5 extender) with final concentration of 30 × 10⁶ spermatozoa / 0.5 mL diluted semen. Diluted semen samples were stored in aliquots at 5ºC up to 216 h (9 days). Diluted semen was evaluated at 0, 2, 4, 6, 24, 48, 72, 96, 120, 144, 168, 192 and 216 h during cooled storage. Fertilizing capacity represented in conception rate% and litter size at birth/doe of stored diluted semen was examined at 0, 24, 72, 120 and 168 h after storage at 5ºC.

Obtained results showed that NZW rabbit semen diluted with extenders having either 10 or 20 mg RJ / 6 mL tris based extender containing 2% bee honey and 18% EY could be used beneficially within a week when stored at 5ºC for artificial insemination (AI), which would facilitate commercial distribution. In conclusion, the addition of bee honey and RJ in rabbit semen diluents improved both semen quality and fertilizing capacity when stored at 5ºC.

Conclusively, the results of the present study suggested that the addition of bee honey and RJ to rabbit semen diluents improved both semen quality and fertilizing capacity during storage at 5ºC. These results revealed that NZW rabbit semen diluted with extenders that contained 10 or 20 mg RJ / 6mL tris-basal extender having 2% bee honey and 18% EY beneficially used within a week when stored at 5ºC for AI. The previous results showed a significant positive effect on the quality of stored semen, which would facilitate commercial distribution.

Key words: Cooling, egg yolk, extender, fertilizing capacity, honey, rabbit, royal jelly, semen.

 

Artificial insemination (A.I.) in rabbits is usually performed using fresh diluted semen (on the day of semen collection) yielded pregnancy rates similar or less than those would be achieved by natural mating (El-Gaafary and Marai, 1994, Morrel, 1995 and Harkness et al., 2010). A.I. has been introduced in industrial rabbitries mainly to improve breeding management (Castellini, 1996).The major target for the rabbit breeder is how to preserve the fertilizing capacity of stored rabbit semen. Extenders are certain ingredients added to the ejaculated semen to sustain and protect the spermatozoa thereby preserving its fertility until they are used for insemination (Geoffrey et al., 1992).The use of honey to partly replace honey in egg yolk based extender (El-Sherbiny, 2013a). Additionally, honey contains high level of metabolizable energy in the form of glucose and fructose (Molan and Russell, 1988 and Al-Waili, 2004).

Royal jelly (RJ) has been used for centuries for its extraordinary properties and health effects (Pavel et al., 2011). Royal jelly is one of the most studied bee products; however, many researches are needed to reveal its biochemical and biological activities. On the other hand, the chemical and bioactive compounds of RJ are not sufficiently known. RJ has a multitude of pharmacological activities (Mărghitas, 2008). The overall composition of RJ is 67% water, 12.5% crude protein (including small amounts of many different amino acids) and 11% simple sugars (monosaccharide), also including a relatively high amount (5%) of fatty acids. It also contains many trace minerals, some enzymes, antibacterial agents, antibiotic components and trace amounts of vitamin C (Graham, 1992). RJ has immunomodulatory activities (Gasic et al., 2007).

El-Sherbiny (2013b) concluded that RJ addition to tris basal extender which contained dimethysulfoxide (DMSO), 2% honey bee and 18% EY improved semen quality and fertility of diluted NZW rabbit semen before and after storage at -195ºC. On buffalo, in vitro study by Abd-Allah (2012) suggested that treating buffalo sperm with 0.4% RJ in combination with heparin is effective not only to induce sperm acrosome reaction but also is effective for in vitro fertilizing capacity of the cryopreserved buffalo spermatozoa. For rabbit semen, fast cooling rates from room temperature to 5°C are likely possible due to the peculiarities of the sperm membrane in this species (high cholesterol: phospholipid ratio) as showed by Mocé and Vicente (2009). Tris-buffer extenders were effective at preserving fertility for two days when stored at 15ºC (Roca et al., 2000). However, attempts are underway to preserve fertility in semen stored for several days. Commercial semen distribution is still of low significance in rabbits due to semen precipitation and energy expenditure movement during storing. Further experimentation (in vitro, in animal’s research) and validation would be needed to prove any useful benefit and action mechanism of native bee honey and RJ and their isolated compounds, as well.

Therefore, the aim of the present study was to investigate the effect of bee honey and/ or RJ addition to tris-buffer extender on the quality and fertilizing capacity of diluted rabbit semen after preservation at 5ºC up to 216 hours.

 

MATERIALS AND METHODS

 

The present study was carried out during winter season 2013/2014 (from December 2013 to February 2014) at the Intensive Rabbit Production Unit, Faculty of Agriculture, Ain Shams University, Cairo, Egypt and a Private Rabbit Farm, Kalioubia, Egypt.

 

Experimental animals: 

A total of twenty sexually mature New Zealand White (NZW) rabbit bucks, aged 18 months with average body weight 4.1 kg, were selected for high quality semen (reaction time < 2 minutes, color white only, volume ≥ 0.2 mL, density creamy and milky only, concentration > 60 million/ml semen, dead-sperm percentage < 20%, and sperm abnormalities <20%). For testing the fertilizing capacity of cooled diluted semen, 460 hybrid nonparous females (5-6 months old) at a private rabbitry, Kalioubia, Egypt, were used in the experiment (10 females each time per each extender). All animals were housed individually in flat deck cages and fed a commercial concentrated pelleted diet according to their physiological and reproductive condition according NRC allowances (NRC, 1977). Fresh water was provided ad libitum.

 

Semen collection and evaluation:

Semen was collected from each buck twice weekly using an artificial vagina (960 ejaculates were used). Two rabbit does were used as a teaser. Immediately after semen collection, gel plug was removed. Only ejaculates with white color and good mass motility (≥3 on a 0–5 scale) were used for semen processing. After collection, semen from ten bucks was pooled together as described by Safaa et al. (2012). The semen quality parameters of rabbit bucks were evaluated (percentages of sperm progressive motility, dead and abnormal spermatozoa) as described by El-Sherbiny (1987) and Madhuri et al. (2012).

 

Semen dilution:

Tris-citric-glucose extender was the basic diluent and had the following composition: 0.25M of Tris buffer (hydroxylmethyl) aminomethan, 0.87M of citric acid monohydrate, 0.47M D (+) glucose, 100000 IU Penicillin and 100 mg streptomycin sulfate (Viudes-de-Castro and Vicente, 1996 and Si et al., 2006), All previous components were dissolved in double distilled water up to 100 mL. Honey was added to the diluent in ascending concentrations (v/v) 0, 1, 2,3, 4, 5, 7.5 and 10% and descending concentrations of EY 20, 19, 18, 17, 16, 15, 12.5 and 10% (v/v), for extenders 1, 2, 3, 4, 5, 6, 7 and 8, respectively. Fresh EY was added just before semen collection (Chen et al., 1989 and El-Kelawy et al., 2012). The bee honey-included EY percentage was 20% (v/v) of diluent according to El-Sherbiny (2013a). RJ was added to tris-buffer extender with 2% bee honey and 18% EY; the concentrations (w/v) of RJ were 10, 20, 30, 40, 50 and 100 mg/6 mL of diluent according to El-Sherbiny (2013b), for extenders 9, 10, 11, 12, 13 and 14, respectively. The dilution rate of pooled semen was one volume of semen to five volume of extender (v/v) at 37^oC. Semen samples were stored in aliquots in a refrigerator at 5ºC up to 216 h (9 days).

 

Semen cooling:

Total sperm-cell concentration (Conc.) was 363.08 million spermatozoa / mL. Hence, Conc. after dilution was 60.51 million spermatozoa / mL. The diluted semen was packed into aliquots (1 mL), then dipped in a glass beaker of water at room temperature (18-22ºC) and stored in a refrigerator at 5ºC. The elapsed time between semen dilution and the beginning of storage didn’t exceed 30 minute.

 

Post cooling examination of diluted semen:

Cooled aliquots were warmed in a water bath at 37°C for 10 seconds (Kashiwazaki et al., 2006). After warming, diluted semen samples were examined for percentage of sperm progressive motility, dead and total abnormal spermatozoa. Diluted semen was examined at 0, 2, 4, 6, 24, 48, 72, 96, 120, 144, 168, 192 and 216 h after cold storage at 5ºC.

Artificial insemination:

Rabbit does chosen for insemination were thought to be sexually receptive (had red color of vulva lips). In order to induce ovulation, females were injected intramuscularly with 0.3 mL receptal (GnRH analogue, 1.26μg of busereline acetate; Intervet, Cairo, Egypt). Diluted semen showed progressive motility less than 50% didn’t use for AI. Then, each doe was inseminated artificially with 0.5 mL diluted semen (containing approximately 30 x 10⁶ sperms) just after GnRH injection (Chen et al., 1989). Pregnancy was detected by trans-abdominal palpation 14 days post-insemination to determine conception rate% (El-Kelawy et al., 2012; EL-Sherbieny et al., 2012 and Safaa et al., 2012). Litter size was determined for each doe directly after kindling. Fertilizing capacity of stored diluted rabbit semen was examined at 0, 24, 72, 120 and 168 h after storage at 5ºC.

 

Statistical analysis:

Data of percentages of sperm progressive motility, dead and abnormal spermatozoa of diluted NZW rabbit semen were analyzed using Two Way Analysis of Variance (ANOVA). Litter size data were analyzed using One Way ANOVA. Whereas, for conception rate trait, Catmod procedure and Chai-square test for homogeneity of variance were performed. All statistical analysis for the different traits was realized using SAS program (SAS, 2011). Differences among experimental groups were tested by Duncan’s Multiple Range test (Duncan, 1955).Two Way ANOVA was according to the following model:

y_ijk = µ + trt_i + time_j + (trt *time)_ij + e_ijk

Where: µ is the overall mean, y_ijk is the observation of the studied trait of k^th animal of i^th trt and j^th time, trt_i is the effect of i^th trt (i = 1, 2, ……….14), Time_ej is the effect of j^th time (j = 1, 2, 3 and 4), (trt*time)_ij is the effect of the interaction between trt and time, e_ijk is the individual error. One way ANOVA was according to the following model:

y_ij = µ + t_i + e_ij

Where: µ is the Overall mean, y_ij is the observation of the studied trait of j^th animal of i^th treatment, t_i is the fixed effect of treatment (i = 1, 2 …., 14), e_ij is the individual error.

 

RESULTS AND DISCUSSION

 

Physical characteristics of diluted NZW rabbit semen:

Estimated physical characteristics of NZW rabbit crude semen before dilution are summarized in Table 1. 

Table 1: Some physical characteristics (Means ± SE) of NZW rabbit semen.

Parameter    Volume         pH             PM%        DS%         AS%               Conc                          (ml)                                                                                      (million/ml)

N                       40               40               40              40                40                   40 Mean               0.6               7.1             89.5            7.4               10.6              363.1    ±SE             ±0.05           ±0.05          ±0.06         ±0.01            ±0.03           ±46.31

PM: Progressive motility; DS: Dead spermatozoa; AS: Abnormal spermatozoa; Conc: Sperm-cell concentration; N: number of evaluated ejaculates.

The effect of bee honey and/ or royal jelly on sperm progressive motility of diluted NZW rabbit semen stored at 5ºC is presented in Table 2. Results showed that the effects of either extender or storage time on percentage of sperm progressive motility (PM) were highly significant (P<0.01). On the other hand, PM was decreased with the increasing of storage time for all extenders used in the present study.

Addition of bee honey to the extender improved PM, but this improvement was concentration dependent. This finding is in agreement with those obtained by El-Sherbiny (2013a). Honey had a significant (P<0.01) positive effect when added in concentrations of 2, 3 and 4% (extenders 3, 4 and 5) compared to Tris-basal extender (extender 1). The inclusion of bee honey as a source of glucose (Molan and Russell, 1988 and Al-Waili, 2004) in EY buffer diluting medium used in the present study supported by the report of Smith et al. (1954) that the addition of small amounts of  glucose to EY buffer increases and prolongs active motility of spermatozoa. This effect could be due to the antibacterial activity of bee honey against some microorganisms resistant to the common antibiotics used in the extender, additionally, honey bee is a good source of glucose and fructose (Molan and Russell, 1988).

In the present study, the inclusion of honey in EY based extenders was found to sustain both sperm motility and livability. However, this effect was found to be dependent on the ratio of honey to EY in the extender. This is in agreement with the findings of Olayemi et al. (2011) on goat semen.

Tris-citric-glucose extender was used as a basal extender in the present study. Roca et al., (2000) mentioned that Tris-citric-glucose extender is effective for dilution and storage of rabbit semen at 15^oC.

El-Gaafary (1994) showed that storage and incubation time decreased (P<0.01) sperm motility and increased (P<0.01) the percentages of spermatozoa with damaged acrosomes and amounts of lactic dehydrogenase

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

released into the extracellular medium. While, the addition of RJ enhanced PM all over the periods of diluted semen examination from 0h to 216 h at 5ºC. The present results proved that the addition of 10 and 20 mg RJ / 6 mL of diluent, showed better PM than the other extenders used. These two RJ concentrations showed not only more than 50% of PM till 168 h of storage at 5ºC, but also maintained PM to 216 h of storage (33 and 22%, respectively).

Results represented in Table 3 showed the effect of bee honey or/ and RJ on percentage of dead spermatozoa (DS). These results indicated that, percentage of DS increased significantly (P<0.01) with advanced time for all extenders examined. The impact of either extender or time on DS was significant at P<0.01; while, bee honey addition in concentrations of 2, 3 and 4% showed DS significantly (P<0.01) lower than the other honey-extenders and Tris-basal extender. Whereas, RJ addition to extender revealed the lowest values (P<0.01) of DS all the storage time at 5ºC, especially in concentrations of 10 and 20 mg RJ / 6 mL of diluent.

Table 4 represents the effect of bee honey or/ and RJ on percentage of abnormal spermatozoa (AS) stored at 5ºC. The results of the present study indicated that both extender and storage time had significant (P<0.01) effect on AS. The lower values obtained for AS were for concentrations of 2, 3 and 4% of honey, but the lowest values were for RJ concentrations of 10 and 20 mg/6 mL of diluent. The results of the present study showed that rabbit semen preservation for 48 hours at 5°C did not affect spermatozoa characteristics, after that, all parameters studied and fertility depreciated. This is in agreement with the results obtained by Echegaray-Torres et al. (2004).

Dead and abnormal percentages of spermatozoa were significantly (P<0.01) increased, while sperm progressive motility decreased significantly (P<0.01) with the increasing of storage time. That may be due to the concentration of toxic substances in the extender during storage, stress from continuous motility of sperm and low energy sources available. Therefore, extenders are suitable for rabbit semen within the first two or three days of storage (Roca et al., 2000 and Echegaray-Torres et al., 2004).

 

Fertilizing capacity of diluted NZW rabbit semen:

Conception rate (CR) and litter size at birth (LS) of the 14 extenders among the different storage time are represented in Table 5. At 0 h, differences among different extenders in both conception rate and litter size at birth were significant (P<0.05). The results showed that insemination of nonparous rabbit does artificially with diluted semen of 2,

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3 and 4% bee honey extenders realized more CR and LS than those inseminated by semen diluted with tris-citric-glucose extender alone at 0 h. On the other hand, RJ extenders showed the highest values of CR and LS compared to other extenders at 0 h. The differences among extenders in CR and LS were highly significant (P<0.01) when examined at 24, 72, 120 and 168 h of storage at 5ºC. Extenders contained less than 5% honey improved fertilizing capacity of diluted NZW rabbit semen until 5^th day of storage at 5ºC (Table 4).

Addition of RJ to extenders significantly (P<0.05) improved fertilizing capacity of diluted rabbit semen stored at 5ºC till 7^th day of storage. Concentrations of 10 and 20 mg RJ/6 mL diluent (extenders 9 and 10) showed the best values of CR and LS compared to the other extenders used in the present study.

Roca et al. (2000) obtained 75.1 % fertility and 7.68 kids/ kindled doe when does inseminated at zero time with rabbit semen diluted in TCG extender. Also, Viudes-de-Castro and Vicente (1997) reported 91% fertility which is close to that obtained in the present study, and 9.4 kids/ kindled doe for the same time and extender type. Roca et al. (2000) suggested that sperm concentration in rabbit insemination dose could be slightly less than 15 million; in the present study, insemination dose had 30 million spermatozoa. Fertility test also, showed that rabbit semen fertilizing capacity deteriorated over storage time more quickly (Echegaray-Torres et al., 2004).

Conclusively, the results of the present study suggested that the addition of bee honey and RJ to rabbit semen diluents improved both semen quality and fertilizing capacity during storage at 5ºC. These results revealed that NZW rabbit semen diluted with extenders that contained 10 or 20 mg RJ / 6mL tris-basal extender having 2% bee honey and 18% EY beneficially used within a week when stored at 5ºC for AI. The previous results showed a significant positive effect on the quality of stored semen, which would facilitate commercial distribution.

 

ACKNOWLEDGEMENT

The author would like to acknowledge the technical assistance of Mr. A.H. Khadr and A.S.S. Baky and Prof. Manal M.A. El-Sayed for her valuable help in Statistical analysis. Also, appreciation was extended to Captain Amro Zaied for providing rabbit does and Prof. A. Bassiouny for providing pure bee honey and royal jelly.

 

 

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تأثیر عسل النحل و الغذاء الملکى على القدرة الإخصائیة للسائل المنوى المخففللأرانب المخزن على درجة حرارة 5ºC  مئویة

 

أحمد محمد الشربینى

قسم الإنتاج الحیوانى – کلیة الزراعة – جامعة عین شمس – القاهرة – مصر

 

تم دراسة تأثیر إضافة عسل و/ أو غذاء ملکات النحل إلى مخفف تریس-ستریک-جلوکوز على الحرکة التقدمیة والحیویة ونسبة الحیوانات المنویة المیتة والشاذة والمقدرة الإخصابیة للسائل المنوى المخفف للأرانب النیوزیلندى البیضاء والمحفوظ على درجة حرارة 5ºC مئویة. تم تجمیع السائل المنوى  و إضافته للمخففات التى أساسها مخفف تریس-ستریک-جلوکوز و التى تحتوى على ترکیزات تصاعدیة من عسل النحل (0 و1 و2 و3 و4 و5 و7.5 و10 %) و ترکیزات تنازلیة (حجم/ حجم) من صفار البیض (20 و19 و 18 و17 و16 و15 و12.5 و10%). تم إضافة غذاء ملکات النحل  لمخفف التریس المحتوى على 2% عسل نحل و18% صفار بیض، و قد کانت ترکیزات غذاء ملکات النحل (وزن/ حجم) 10 و20 و30 و40 و50 و100 مجم / 6 مللیلتر من المخفف. نسبة التخفیف کانت 1: 6 (1 سائل منوی + 5 مخفف) بحیث یحتوى کل 0,5 مللیلتر من السائل المنوی المخفف على 30ملیون حیوان منوى. تم حفظ عینات السائل المنوی المخفف فی عبوات محکمة على درجة حرارة 5ºC مئویة لفترات تصل إلى 216 ساعة (9 أیام)، تم تقییم السائل المنوی المخفف قبل الحفظ (الوقت 0) ثم بعد 2 و4 و6 و24 و48 و72 و96 و120 و144 و168 و192 و216 ساعة من الحفظ المبرد. تم تقدیر المقدرة الإخصابیة (معدل الحمل % وعدد الخلفة عند المیلاد لکل أم) بعد 0 و24 و72 و120 و168 ساعة من التخزین على درجة حرارة 5ºC مئویة.

أظهرت النتائج أن السائل المنوى المخفف للأرانب النیوزیلندی البیضاء والمضاف إلیه 10 أو 20 مجم غذاء ملکات النحل/ 6 مللیلتر من مخفف التریس المحتوى على 2% عسل نحل و18% صفار بیض، یمکن استخدامه بأمان خلال فترة أسبوع من الحفظ المبرد على درجة حرارة 5ºC مئویة لغرض التلقیح الإصطناعى، والذی قد یسهل عملیة النقل والتداول التجاری للسائل المنوى المخفف.

التوصیة: یتضح من نتائج هذه الدراسة أن إضافة عسل النحل وغذاء ملکاته إلى مخففات سائل منوی الأرانب یمکن أن یحسن کلا من الخصائص النوعیة للسائل المنوی والمقدرة الإخصابیة له عند حفظه وتخزینه على درجة حرارة الثلاجة ( 5ºC مئویة).

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