VIABILITY; ENZYMATIC ACTIVITY AND FERTILIZING ABILITY OF RABBIT SEMEN AS AFFECTED BY INJECTION OF GIBBERELLIC AND BORIC ACID OR BOTH

Safaa, A. Barakat;  Amal, M. Hekal and T.S.T. Seleem

Department of Rabbit; Turkey and Water Fowl Breeding Research, Animal Production Research Institute, Agricultural Research Center, Dokki, Giza, Egypt.

E.mail of corresponding author: safaa_ataya@yahoo.com

A total number of 244 New-Zealand White (NZW) rabbits (48 sexually mature bucks 9 months of age, and 196 nonparous does 6 months of age) were used in the present study. The study lasted 4 months and was designed to estimate fertilizing ability of bucks and fertility traits of does as affected by injection of Gibberellic and Boric acid or both. Rabbits were divided into four experimental comparable groups (12 bucks and 49 does each). Animals in 1^st group were subcutaneously injected with 1 ml saline solution (NaCL) 0.9% and served as a control group, while the rabbits in 2^nd, 3^rd and 4^th groups were injected subcutaneously with 1 mg Gibberellic acid, 2.5 mg Boric acid and mixture of 1 mg Gibberellic and 2.5 mg Boric acids, respectively. The present investigation included two experiments. The first experiment (in vitro) was designed to investigate semen quality and storagability; spermatozoa enzymatic activity and sperm penetration into estrus cow cervical mucus. The second experiment (in vivo) was designed to estimate fertility traitsof does artificially inseminated by bucks treated with the same treatments.  

Results obtained showed that, Diluted semen of bucks injected with Gibberellic and Boric acid or both, recorded higher(P ≤ 0.01)  sperm motility and storagability than control group, during conservation at both incubation and refrigeration temperatures. Dead and abnormal spermatozoa and acrosomal damages percentages of diluted semen recorded significant (P ≤ 0.01) decrease. Spermatozoa enzymatic activityt, decreased significantly (P ≤ 0.01) and sperm penetration into estrus cow cervical mucus significantly (P ≤ 0.01) increased. Physical semen quality decreased significantly (P≤0.05) with progress of conservation time at incubation condition (37 °C)up to 6 hours, or at  refrigeration temperature (4-6 °C) up to 3 days. Fertility traits of NZW rabbit does were significantly (P≤0.0.5 or 0.01) better by injecting Gibberellic and Boric acid or both.

Conclusively, it could be concluded that, injecting rabbits with mixture of Gibberellic and Boric acids improved semen quality and storagability of bucks, and fertility traits of does in comparison with administration of each acid alone.

  Key words: Rabbit, semen, Gibberellic, Boric, incubation, enzymes, fertility.

Animal protein deficiency is a very serious problem in developing countries as Egypt. This isn’t only due to the small number of existing farm animals as compared to the rapid growth of human population, but also due to the low productive and reproductive capabilities of local farm animals (Rowida Riad and Seleem, 2007).

F.A.O. (1987) revealed that, rabbit farming is encouraged to minimize the gab between demand and supply of animal protein. Rabbits have their advantages including, short life cycle, small body size, good quality meat with high nutritional-biological value and low cholesterol content and easy to digest, short generation interval and high reproductive potential and prolificacy (Gad and Seleem, 1999). Moreover, rabbits characterized by good ability to utilize diets contained ingredients of agricultural wastes and food industrial by-products which aren’t used directly for human consumption. As well as, rabbit feeding and management considered too reasonable in cost compared with any other kind of farm animals (Daader and Seleem, 1999) that make them suitable as protein-producing small animals. So, rabbits industry had gradually increased attention in the last decade in developing countries for meat production (Seleem, 2005).

Recently, artificial insemination (AI) is widely employed and most suitable for both narrow and large commercial Rabbitries (Lavara et. al., 2005). So, the storagability of rabbit semen for the longest possible time is essential purpose.

Many factors affect seminal traits (Boiti et. al., 2005) and thus it is crucial to define suitable protocols to improve spermatozoa characteristics (Brun et. al., 2002). Hence, many attempts have been applied to use some stimulators in order to maximize and prolong survivability of spermatozoa and fertilizing ability during storage of rabbit semen at different temperatures. The stimulators included hormones (Seleem, 2003) and like; hormones (Abd-El-Kariem et. al., 1998) stimulating hormones (Daader et. al., 1997 and 2002); chemical substances (Marwa, 2002) and herbs aqueous extract (Seleem et. al. 2007).

Gibberellic acid is essential acid regulates various physiological processes (Alkhiat et. al., 1981). Gawienowski and Chatterijee (1980) showed that Gibberellic acid is considered estrogenic hormone-like actions resulted improvement in libido and semen quality of males and increasing maternal ability of females.  Kamel et al. (2009) used Gibberellic acid at 400 µg/ kg body weight/ week and revealed its importance in stimulating and supporting spermatogenesis and sex accessory glands. It caused an improvement in sperm enzymatic activity which represented in significantly decrease in alanin aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes and significantly increase in alkaline phosphatase activity (ALP) and acid phosphatase activity (ACP) enzymes in seminal plasma. The last authors recorded a significant increase in serum testosterone hormone concentration due to Gibberellic acid administration.

Heindel et. al. (1994) and Catherine et al. (1996) recorded a significant increase in litter size and weight at birth and at weaning, milk quality and quantity and pre-weaning mortality rates of rabbit does due to 125 µg/ kg/ day Boric Acid treatment. They added, that Boric acid display an essential role in regulating osmotic pressure and pH of seminal plasma which resulted in improving fertilizing ability of rabbit males.

Bacterial contamination of semen intended for artificial insemination was reported to have a deleterious effect on sperm quality, longevity and ultimately fertility. Inclusion of antibiotics in semen diluents is to reduce those detrimental effects (Poolperm, 2001). The frequent use of antibiotics randomly in Rabbitries either for animals or semen treating may cause antibiotics-resistance strains of bacteria (Rowida Riad, 2003). So, great attention must be given to use some other materials and compounds instate of antibiotics in rabbit industry. Gibberellic and Boric acids have antibacterial effects and they may display an essential role indirectly in semen preservation for long periods (Fadila Easa et. al., 2013).

Therefore, the aim of the present work was to study viability; enzymatic activity and fertilizing ability of NZW rabbit semen, and fertility traits of NZW rabbit does as affected by injection of Gibberellic and Boric acid or both.

MATERIALS AND METHODS

Animals and management

The field part of the present study was carried out in an industrial Rabbitry, Sakarah city, Giza province, Egypt. whereas the laboratory work was conducted in Animal Production Research Institute, Agriculture Research Center, Giza, Egypt. The study lasted four months.

A total number of 244 New-Zealand White (NZW) rabbits (48 sexually mature bucks nine months of age with 3115 ± 35 g average body weight, and 196 nonparous does six months of age with 3050 ± 35 g average body weight) were used in the present work. All animals were fed ad libitum a commercial pelleted diet according to NRC (1977) recommendations. The ingredients and chemical composition of the pelleted ration fed to rabbits, during the experimental period were shown in Table 1.

Animals were healthy and clinically free of external and internal parasites. The animals were kept under the same managerial and hygienic conditions. 

The experimental design

 Animals were divided into four experimental comparable homogeneous groups (12 bucks and 49 does each). Rabbits in the1^st group were subcutaneously injected with 1 ml saline solution (NaCL, 0.9%) and served as a control group. While, the rabbits in the 2^nd, 3^rd and 4^th groups were injected subcutaneously with 1 mg  Gibberellic acid, 2.5 mg Boric acid and mixture of 1 mg  Gibberellic and 2.5 mg Boric acids, respectively. The volume of each injecting dose was 1 ml at all treatments. Rabbit injection was started at 6 weeks before semen collection and at weekly intervals during the whole experimental period. The present study included two experiments. The first experiment (in vitro) work was designed to determine semen quality and storagability; spermatozoa enzymatic activity and sperm penetration into estrus cow cervical mucus. The second experiment (in vivo) work was planned to evaluate rates of conception, abortion and kindling, litter size and weight and bunny weight at birth and at weaning, in addition to pre-weaning mortality rate.  

Diluted semen preservation

The ejaculated semen of each buck within all treated groups was individually evaluated microscopically. Semen samples were pooled and diluted with sodium citrate diluent (2.9 di-sodium citrate + 1.25 gm lactose + 0.04 gm citric acid anhydrous + 5.0 ml egg yolk + 50000 IU sodium penicillin + 50000 µg streptomycin sulfate and completed up to 100 ml with distilled water) at 1: 5 dilution rate. The diluted semen was divided into two equal parts; the first was chilled at refrigeration temperature (4 - 6 ^oC) for up to 3 days, and the second was incubated at 37 ^oC for up to 6 hours. Percentages of advanced sperm motility, spermatozoa storagability, abnormal and dead spermatozoa and acrosomal damages were recorded at different stages of preservation according to Boiti et. al. (2005) and Castellini et. al. (2006).     

                   Final advanced sperm motility (after 6 hours)

Storage ability = ---------------------------------------------------------------X 100

                   Initial advanced sperm motility (at zero time)        

Table 1. The ingredients and chemical composition of the pellet ration fed to rabbits, during the experimental period.    

** Calculated according to NRC (1977) for rabbits.

Spermatozoa enzymatic activity

Diluted semen samples ejaculated by treated NZW rabbit bucks were centrifuged at 6000 r.p.m. for 20 minutes before removal of the supernatant and used for enzymatic assay. The activities of aspartate aminotransferase (AST) and alanine aminotranferase (ALT) were estimated according to Reitman and Frankel (1957) and Gella et al. (1985). Acid phosphatase (ACP) and alkaline phosphatase (ALP) enzymes were determined colorimetrically according to Graham and Pace (1967).

Sperm penetration into estrus cow cervical mucus

Sperm penetration into estrus cow cervical mucus of diluted NZW rabbit semen was assessed during incubation condition at 37 ⁰C for 6 hours as described by Rowida Riad et al. (2009) and Seleem et al.  (2009).

Fertility traits

196 nonoparous NZW rabbit does were artificially inseminated using diluted semen ejaculated by corresponding experimental groups of rabbit bucks. The artificial insemination was carried out as described by Boiti et. al. (2005). Palpation of all rabbit does was executed at day 12 post insemination to determine pregnancy and conception rates. At kindling, conception, abortion, kindling rates, litter size, weight at birth and at weaning values were  recorded. Pre weaning mortality rates of NZW rabbit does were  estimated.

Statistical analyses

Data were subjected to analysis of variance according to Snedecor and Cochran (1982) using the General Linear Model Program of SAS (2001). Duncan^'s new multiple range tests were used to test the significance of the differences among means (Duncan, 1955). Data presented as percentages were transformed to the corresponding arcsine values (Warren and Gregory, 2005) before being statistically analyzed. Conception, abortion and kindling rates were analyzed using the Contingency Tables according to Everitt (1977).

RESULTS AND DISCUSSION

Diluted semen preservation

Data presented in Tables 2 & 3 showed that, diluted semen of NZW rabbit bucks injected with Gibberellic and Boric acid or both, recorded higher (P ≤ 0.01) sperm motility and storagability percentages than those of NaCl injected bucks (control), during conservation at both incubation and refrigeration temperatures. Percentages of dead spermatozoa; sperm abnormalities and acrosomal damages of diluted NZW rabbit semen recorded significant (P ≤ 0.01) decrease due to injection with Gibberellic and Boric acid or both.

Percentages of sperm motility in preservation time of diluted semen in all treated groups were decreased significantly (P≤0.05) associated with significant increase (P≤0.05) in percentage of dead spermatozoa, sperm abnormalities and acrosomal damages. This trend is similar to the results obtained by Rowida Riad et al. (2004) and Seleem et al. (2007). The decrease recorded in the percentages of sperm motility may be due to the decrease in content of adenosine triphosphate resulting in inactivation of spermatozoa (Zeidan et. al., 2008). This trend might attributed to an increase in lactic acid accumulated by time caused by the sperm anaerobic metabolism leading to changes in both osmotic pressure and pH in the media exerting deleterious effect on the sperm cells (Seleem, 1996 and Rowida Riad, 2003).

Spermatozoa enzymatic activity

Data presented in Table 4 showed that, values indicated spermatozoa enzymatic activity of NZW rabbit bucks represented in leakage of AST; ALT; ACP and ALP into intracellular media, decreased significantly (P ≤ 0.01) due to Gibberellic and Boric acid injection. Similar results were studied by Hassan and Mervat Ghazal (2013) used red algae.

The increase in leakage of the intracellular AST; ALT; ACP and ALP enzymes into the extra cellular medium may reflect the breakdown of the sperm cellular membrane (Seleem, 2005 and Zeidan et al., 2008). Whereas leakage of these enzymes decreased significantly (P≤ 0.05) by injecting Gibberellic and Boric acid or both. These results emphasize that, Gibberellic and Boric acids are able to prevent the increase of the spermatozoa enzymatic activity in the diluted rabbit semen and consequently decreasing activity of AST; ALT; ACP and ALP enzymes, which were paralleled to the Gibberellic and Boric acid or both injection.

The cell membrane integrity is the key factor for explaining such a trend. Continuous increase in leakage of intracellular AST, ALT, ACP and ALP enzymes into the extra cellular medium during incubation time may reflect the breakdown of the sperm cellular membrane (Seleem et al., 2007 and Zeidan et al., 2008).

Sperm penetration into estrus cow cervical mucus

Table 5 showed that,sperm penetration into estrus cow cervical mucus significantly (P ≤ 0.01) increased due to Gibberellic and Boric acid or both injection.Incubated semen recorded a significant increase (P≤0.05) in sperm penetration into estrus cow cervical mucus with advancement of incubation period. These results are comparable with those obtained by Daader et al. (2002) and Seleem et al. (2008).   

The significant increase in ability of spermatozoa to penetrate estrus cow cervical mucus may be attributed to the beneficial effect of Gibberellic and Boric acid or both injection on increasing advanced sperm motility and semen quality in the reactivated media (Tables 2 & 3) and consequently increasing sperm penetration into cervical mucus.

Fertility traits of rabbit does

Data presented in Table 6 showed that,fertility traits of NZW rabbit does were significantly (P≤0.05 or 0.01) better by injecting Gibberellic and Boric acid or both, when inseminated artificially by using bucks received the same trend. These findings may attribute to the improvement of semen quality of rabbit bucks injected with Gibberellic and Boric acid or both.

It's apparent clearly from obtained results that, the reproductive efficiency of both rabbit bucks and does injected with mixture of (1 mg Gibberellic + 2.5 mg Boric acids) were better than those injected with only 2.5 mg Boric or 1 mg Gibberellic acids. These results may be due to the combined effect of Gibberellic and Boric acids (Fadila Easa et. al., 2013).

Alkhiat et. al., (1981) emphasized the essential role of Gibberellic acid in regulating various reproductive processes. Gawienowski and Chatterijee (1980) reported that Gibberellic acid is considered estrogenic hormone-like

Table 4. Enzymatic semen activity of NZW rabbit bucks as affected by Gibberellic and Boric acid or both injection (Means ± SE).

Means bearing different letter superscripts (a,b,c) within the same row are significantly  (P ≤ 0.05 or 0.01) different.

Table 5. Sperm penetration into estrus cow cervical mucus (mm/ Hrs.) of NZW rabbit bucks as affected by Gibberellic and Boric acid or both injection (Means ± SE).

Means bearing different letter superscripts (a,b,c,d) within the same row or (A,B,C,D) within the same column of each treatment are significantly  (P ≤ 0.05 or 0.01) different.

actions resulted in improvement in sexual desire (libido) and all semen parameters of rabbit bucks.  Kamel et. al. (2009) used Gibberellic acid at 400 µg /kg body weight weekly to stimulate and support spermatogenesis and sex accessory glands resulted an improvement in spermatozoa longevity and sperm enzymatic activity.

Furthermore, Heindel et. al. (1994); Catherine et. al. (1996) and Fadila Easa et. al. (2013) recorded a significant improvement in semen quality, during semen preservation due to Boric acid treatment. She added that, Boric acid display an essential role in regulating osmotic pressure and pH value of seminal plasma lead to improve fertilizing ability of males.

Conclusively, it could be concluded that, the combined effect by injecting rabbit bucks with mixture of (1 mg Gibberellic + 2.5 mg Boric acids) was more pronounced and effective than using each acid alone.This additive and beneficial effect is evidenced by prolonging the functional activity and

Table 6. Fertility traits and total pre-weaning mortality rates of NZW rabbit does artificially inseminated using semen of bucks injected with Gibberellic and Boric acid or both (Means ± SE).   

Means bearing different letter superscripts (a,b,c,d) within the same row are significantly 

(P ≤ 0.05 or 0.01) different.

maintaining the cell membrane integrity of spermatozoa along with an improving in the sperm motility penetration into cervical mucus during semen conservation. In addition, fertility traits of rabbit does inseminated artificially could be improved by injecting both bucks and does this mixture.

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تأثیر الحقن بحامضى الجبریلیک أو البوریک أو الإثنین معاً على الحیویة والنشاط الإنزیمى و المقدرة الإخصابیة للسائل المنوى فى الأرانب

صفاء عطایا برکات، أمل مغاورى هیکل،  طارق سلیمان توفیق سلیم

قسم بحوث تربیة الأرانب والرومى والطیور المائیة، معهد بحوث الإنتاج الحیوانى، مرکز البحوث الزراعیة، الدقى، جیزه، مصر.

أستخدم فى هذه الدراسة  244 أرنب نیوزیلندى أبیض (48 ذکر ناضج جنسیاً عمر 9 أشهر و  196إناث فى الموسم الأول عمر 6 أشهر). إستمرت الدراسة أربعة أشهر، وتهدف الدراسة إلى تقییم تأثیر الحقن بحامضى الجبریلیک أو البوریک أو مخلوط منهما على المقدرة الإخصابیة للذکور ومعدلات خصوبة الإناث فى الأرانب. قسمت الأرانب إلى أربعة مجموعات تجریبیة متجانسة (12 ذکر، و49 أم فى کل مجموعة). حقنت أرانب المجموعة الأولى تحت الجلد بـ 1 مل محلول ملحى (0,9 % کلورید الصودیوم)، وإستخدمت کمجموعة مقارنة، بینما أرانب المجموعات التجریبیة الثانیة، والثالثة، والرابعة، حقنت تحت الجلد بـ 1 ملجم حامض الجبریلیک، أو2,5 ملجم حامض البوریک، أو خلیط من 1 مجم حامض الجبریلیک + 2,5 ملجم  من حامض البوریک ، على الترتیب.

إشتملت الدراسة على تجربتین أساسیتین، صممت التجربة الأولى (دراسة معملیة) لتقییم تأثیر حقن ذکور الأرانب بحامضى الجبریلیک أو البوریک أو مخلوط منهما على  کل من جودة السائل المنوى ومقدرته التخزینیة، والنشاط الإنزیمى للحیوانات المنویة، ومقدرة الحیوانات المنویة على النفاذ فى المخاط الرحمى. أما التجربة الثانیة (دراسة حقلیة) تهدف إلى دراسة تأثیر حقن إناث الأرانب بحامضى الجبریلیک أو البوریک أو ةمخلوط منهما على مقاییس الخصوبة للإناث ، ولقحت الإناث اصطناعیاً باستخدام الذکور المعامله بنفس معاملة الأمهات.

أوضحت النتائج أن السائل المنوى المخفف للذکور المحقونة بحامضى الجبریلیک أو البوریک أو مخلوط منهما کان أفضل معنویاً (عند مستوى 1%) فى نسبتى الحرکة التقدمیة للحیوانات المنویة ومقدرتها التخزینیة، مقارنة بالسائل المنوى للمجموعة المقارنة ، خلال الحفظ فى الصورة السائلة على درجتى حرارة التحضین والثلاجة. کانت النسب المئویة لکل من الحیوانات المنویة المیتة والشاذة وشواذ الأکروسوم فى السائل المنوى المخفف أقل معنویاً (عند مستوى 1%). کانت معدلات خصوبة إناث الأرانب النیوزیلندى البیضاء أفضل معنویاً (عند مستوى 5% أو 1%) نتیجة للحقن بحامضى الجبریلیک أو البوریک أوة مخلوط منهما.

إنخفض معنویاً (عند مستوى 1%) النشاط الإنزیمى للحیوانات المنویة ، بینما سجلت مقدرة الحیوانات المنویة على النفاذ فى مخاط عنق الرحم زیادة معنویة (عند مستوى 1%). انخفضت جودة وخصائص السائل المنوى الطبیعیة معنویاً (عند مستوى 5%) بتقدم زمن حفظ السائل المنوى المخفف على درجة حرارة التحضین (37°م) حتی 6 ساعات ، أو درجة حرارة الثلاجة (4-6 °م) لمدة ثلاثة أیام.

التوصیة:  ویمکن أن نستخلص من هذه الدراسة فى مجملها أن حقن الأرانب بحامضى الجبریلیک والبوریک معا أدى إلى تحسین فى صفات السائل المنوى ومقدرته التخزینیة بالنسبة للذکور، وکذلک أدى إلى زیادة فى معدلات خصوبة الإناث.