EFFECT OF GIBBERELLIC AND BORIC ACID AND BOTH INJECTION ON REPRODUCTIVE EFFICIENCY OF RABBITS

Amal M. Hekal; Fadila, M. Easa; and Safaa, A. Barakat

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

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

The present study was designed to evaluate reproductive efficiency of rabbits as affected by Gibberellic and Boric acid injection. Two hundred and forty four New-Zealand White (NZW) rabbits (48 sexually puberty males of 3 months of age, and 196 females of 2.5 months of age) were used in the present study.

Rabbits were divided into four experimental comparable groups (12 males and 49 females in 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 study included two experiments. The first experiment was designed to study the effect of Gibberellic and Boric acid injection on some parameters indicated fertilizing ability of NZW rabbit bucks. The second experiment was planned to evaluate fertility traits of NZW rabbit does as affected by Gibberellic and Boric acid and both injection and mated naturally by bucks treated with the same treatments.  

Results obtained indicated that, absolute and relative weights of each of testes; epididymis; sexual accessory glands; pituitary gland; scrotal circumference and testicular index of NZW rabbit males injected with Gibberellic and Boric acid, were significantly (P ≤ 0.01) higher than those of NaCl injected males. Age at first mating of NZW rabbit males decreased significantly (P ≤ 0.05); while weight at first mating and mating activity significantly (P ≤ 0.05) increased. Improved significantly (P ≤ 0.01) libido and physical semen characteristics, due to Gibberellic and Boric acid injection.

Fertility traits of NZW rabbit does were significantly (P≤0.05 or 0.01) better by injecting Gibberellic and Boric acid. Milk yield and total pre-weaning mortality rates of NZW rabbit does from birth up to 35 days, and milk composition were significantly better by injecting Gibberellic and Boric acid.

 Generally, it could be concluded that, injecting rabbits with Gibberellic and Boric acids improved fertilizing ability of bucks, and fertility traits of does.

Key words: Rabbit; Gibberellic; Boric; reproduction; semen; fertility; milk.

Developing countries included Egypt are often characterized by animal protein deficiency. This is not only due to the small number of existing farm animals as compared to rapid growth of human population, but also due to the low productive and reproductive capabilities of these animals (Seleem et. al., 2011).

The increase in animal protein production may come from short-life cycle animals kept by the small holder farmers such as rabbits (Galal and Khalil, 1994). Rabbit farming is encouraged to minimize the gab between demand and supply of animal protein (F.A.O. 1987). So, rabbits had gradually increased attention in the last decade in Egypt for meat production (Rashwan and Marai, 2001 and Seleem, 2003).

In Egypt, rabbit industry is currently facing hard time owing to high cost incurred on feed. So, establishing new Rabbitaries and expansion of that type of animal production project become too limited resulted a decrease in animal protein (Seleem et al., 2011). Several researches have conducted to reduce the high cost of rabbits feed. Such investigations included various feed additives such vitamins; enzymes; minerals; antibiotics; natural herbs; etc. (Boulos et al., 1992; Seleem et al., 2006; 2007 a& b; El-Kholy et al., 2008; Hanna et al., 2008 and Seleem et al., 2008 a& b). Several other kinds of products now available especially after the ban on using antibiotic growth promoter (Marzo, 2001).

Several attempts have been carried out to promote and stimulate rabbits productivity and reproduction using Gibberellic and Boric acids (Fadila Easa et. al.,2013 and Safaa Barakat et al., 2013). This may be due to its ability to regulates various physiological processes  (Alkhiat et al., 1981). Safaa Barakat et al. (2013) concluded that, injecting rabbits with Gibberellic and Boric acids improved semen quality and storagebility of bucks, and fertility traits of does. Fadila Easa et al. (2013) added that, injecting rabbit bucks with Gibberellic and Boric acids improved their semen quality, during semen conservation at incubation condition.

Gibberellic acid is considered estrogenic hormone-like actions resulted improvement in libido and semen quality of males and increasing maternal ability of females (Gawienowski and Chatterijee, 1980).  Kamel et. al. (2009) used Gibberellic acid at 400 µg/ kg body weight/ week and revealed stimulating and supporting spermatogenesis and sex accessory glands. It causes an improvement in sperm enzymatic activities represented in significant decrease in alanin aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes and significant increase in alkaline phosphatase activity (ALP) and acid phosphatase activity (ACP) enzymes in seminal plasma. The last author recorded a significant increase in serum testosterone hormone concentration due to Gibberellic Acid.

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, Boric Acid display an essential role in regulating osmotic pressure and PH of seminal plasma resulted in improving fertilizing ability of males.

Therefore, the aim of this study was to detect some reproductive capabilities of mature NZW rabbits as affected by Gibberellic and Boric acid injection.

MATERIALS AND METHODS

Animals and general management

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

A total number of 244 New-Zealand White (NZW) rabbits (48 sexually puberty males of 3 months of age and 2450 ± 25 gm body weight, and 196 females of 2.5 months of age and 2150 ± 25 gm body weight) were used in the present work. All animals were fed ad libitum a commercial pelleted diet covered their different physiological status according to NRC (1977) recommendations. The ingredients and chemical composition of the pelleted ration fed to rabbits, during the experimental period was shown in Table 1.

The experimental work

The present research was planned to study fertilizing ability and fertility traits of NZW rabbit bucks and does, respectively, as influenced by Gibberellic and Boric acid injection. All rabbits were divided into four

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.

experimental comparable homogeneous groups (12 males and 49 females in each). Rabbits in the 1^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 a mixture of 1 mg  Gibberellic and 2.5 mg Boric acids, respectively. The volume of each injecting dose was 1ml/rabbit/week, during experimental period. The present work included two experiments as follow:-

The 1^st experiment was designed to study the effect of Gibberellic and Boric acid injection on some parameters indicative of fertilizing ability of NZW rabbit bucks. Parameters these included: absolute and relative weights of each of testes; epididymis; sexual accessory glands; pituitary gland; scrotal circumference and testicular index; age and weight at first mating; mating activity; libido and physical semen characteristics( represented in values of each of semen-ejaculate volume; mass and advanced sperm motility; dead and abnormal spermatozoa and acrosomal damages (%) and sperm-cell concentration per ml and per ejaculate), as well as, testosterone concentration.

At 5 months age, three rabbit bucks from each group within the 1^st experiment were randomly taken for slaughter after being fasted for 12 hours (Abd El-Monem, 1995). After complete bleeding, testes; epididymis; sexual-accessory glands and pituitary gland were weighed after the rabbits were slaughtered and relative weights of those organs were calculated according to the following formulae: relative weight of organ = organ weight/ alive body weight (Fu-Chang et al., 2004). Scrotal circumference which was measured by the method described by Boiti et al. (2005). Testicular index (length × width × depth) was calculated in cubic centimeters as recorded by Castellini et al. (2006).

Mating activity (frequency of mating within 15 minutes) of mature rabbit bucks was determined using sexually receptive does. At maturation, libido (sexual desire) was assessed in terms of reaction time in seconds estimated from the time of introducing doe to the buck until the buck started to mount (Castellini et. al., 2006). Semen was collected artificially twice a week individually from each buck within each treated group, by means of an artificial vagina as described by Boiti et. al. (2005). Immediately after collection, semen ejaculate volume (mL); mass motility (score); advanced sperm motility (%); dead and abnormal spermatozoa (%); sperm-cell concentration (N×10⁶/ ml) and total-sperm output (N×10⁶/ ejaculate) were estimated according to Salisbury    et al. (1978); Boiti et al. (2005) and Castellini et al. (2006). Acrosomal damages were determined by using a Giemsa stain procedure as described by Watson (1975).

Age and weight of rabbit males at first mating were recorded. Regarding sexual hormones, during the 5^th month of age, blood samples were taken from the marginal ear vein of six rabbit bucks and does within each experimental group weekly. Blood serum testosterone concentration of bucks and estradiol 17_2α and progesterone levels of does were determined using RIA Kits (Immunotech, A Coulter Co., France) according to the manufacturer information.

2^nd experiment was planned to evaluate fertility traits of NZW rabbit does as affected by Gibberellic and Boric acid injection and mated naturally by bucks received the same treatment and within the same treated group of doe. The parameters carried out were rates of each of conception, abortion, and kindling and values of each of litter size and weight at birth and at weaning, as well as, bunny weight at birth and at weaning; milk yield and total pre-weaning mortality rates from birth up to weaning on 35 days, and milk composition (protein; fat; lactose and ash), on the 21^st day (the peak of milk production).

Fertility traits

To determine the fertility traits, 196 non-parous NZW rabbit does in four experimental groups (49 in each) were naturally mated using the corresponding experimental groups of rabbit bucks. Palpation of all rabbit does was carried out 12 days post mating to determine pregnancy. At kindling, abortion and kindling rates and litter size and weight at birth values were recorded. Pre weaning mortality rates and milk yield per doe were estimated also during the suckling period. Milk yield was estimated after deprivation of pups from suckling their mothers at 8 a.m. daily, then the doe and her pups were weighed before and after suckling, the average of decrease and increase in doe and pup's weight, respectively, was used as the doe milk yield.

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

Reproductive parameter of bucks

Data presented in Tables 2 and 3 indicated that, injecting NZW rabbit males with combination of (1 mg Gibberellic + 2.5 mg Boric acids); 2.5 mg Boric acid and  1 mg Gibberellic acid significantly (P≤0.05) improved in descending order, respectively, their absolute and relative weights of each of testes; epididymis; sexual accessory glands; pituitary gland; scrotal circumference and testicular index than those of NaCl injected males. Age at first mating of NZW rabbit males decreased significantly (P ≤ 0.05); while weight at first mating and mating activity significantly (P ≤ 0.05) increased, due to Gibberellic and Boric acid injection.

Table 2. Some parameters indicated fertilizing ability of NZW rabbit

               bucks as affected by Gibberellic and Boric acid injection

               (Means ± SE).

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

 (P ≤ 0.05 or 0.01) different.

Table 3. Age and weight at first mating and mating activity of NZW rabbit

               bucks as affected by Gibberellic and Boric acid 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 4 showed that, values of each of libido and physical semen characteristics represented by each of semen-ejaculate volume; mass and advanced sperm motility; dead and abnormal spermatozoa and acrosomal damages (%) and sperm-cell concentration per ml and per ejaculate of NZW rabbit bucks significantly (P≤0.05) improved in descending order due to injection the combination of (1 mg Gibberellic + 2.5 mg Boric acids); 2.5 mg Boric acid and 1 mg Gibberellic acid, respectively.

Sexual hormones

Male and female sexual hormones of NZW rabbits represented by concentration of each of (testosterone; estradiol 17_2α and progestrone), recorded an increase significant (P ≤ 0.01) in descending order, due to the treatment with combination of (1 mg Gibberellic + 2.5 mg Boric acids); 2.5 mg Boric acid and 1 mg Gibberellic acid, respectively (Table 5).

These results are partially comparable with those recorded by Metwally et. al. (2002) who used probiotic; Seleem et. al. (2007c) with prebiotic and Seleem et. al. (2011) used a combination of prebiotic and probiotic. Furthermore Heindel et. al. (1994); Catherine et. al. (1996); Fadila Easa et. al. (2013) and Safaa Barakat et. al. (2013) recorded a significant improvement in rabbit semen quality due to Gibberellic and Boric acids treatment.  Fadila Easa et. al. (2013) and Safaa Barakat et. al. (2013) reported a positive correlation between (Gibberellic and Boric acids) injection and reproductive capabilities of rabbit bucks and does. Safaa Barakat et. al. (2013) recorded a significant decrease in sperm enzymatic activity in seminal plasma of rabbit bucks injected with Gibberellic and Boric acids. They added that, there are strongly positive correlation between

AST activity in seminal plasma and sperm concentration; live sperm percent; sperm motility; semen volume and fertilizing ability of semen. They reported also that, AST; ALT; ACP and ALP enzymes play an essential role in sperm metabolism through its involvement in the vital cellular process. Kamel et. al. (2009) and Safaa Barakat et. al. (2013) indicated that, the activity of some important enzymes may be improved due to Gibberellic and Boric acids injection, which could result in development and maturation of sperm in the testes.

Some doe traits

Data presented in Tables 6 , 7 and 8 showed that, NZW rabbit does injected with combination of  (1 mg Gibberellic + 2.5 mg Boric acids); 2.5 mg Boric acid and 1 mg Gibberellic acid and naturally mated by bucks treated with the same treatment, recorded conception; abortion and kindling rates; litter size and weight at birth and at weaning; bunny weight at birth

Table 5.Sexual hormones concentration of NZW rabbit bucks as affected by Gibberellic and Boric acid injection (Means ± SE).

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

and at weaning, as well as, milk yield and composition, in addition to pre-weaning mortality rate significantly (P≤0.05 or 0.01) better, and in descending order, than of those injected with saline solution (control group).

These results are in close agreement  with those of Safaa Barakat    et al. (2013) using Gibberellic and Boric acids and artificial insemination. The same trends were recorded previously by Metwally et. al. (2002); Seleem et al. (2007c) and Seleem et al. (2011).

In this respect, Heindel et al. (1994); Catherine et al. (1996) and Kamel et al. (2009) observed that, the Gibberellic and Boric acids lead to increase feed efficiency in rabbits. This may lead to increased milk secretion and its yield in treated rabbits. Beside that, the increase milk production may be due to increase in litter size at birth, where there was a positive correlation between the litter size at birth and milk yield (Lebas et. al., 1997 and Rommers et al., 2001).

Alkhiat et al., (1981) emphasized the essential role of Gibberellic acid in regulating various reproductive processes. Fadila Easa et. al., (2013) and Safaa Barakat et al., (2013) recorded a significant improvement in rabbit reproductivity due to Gibberellic and Boric acids injection. Gawienowski and Chatterijee (1980) found Gibberellic acid is considered as estrogenic hormone-like actions resulted improvement in sexual desire (libido) and all semen parameters of rabbit bucks.  Kamel et al. (2009) used Gibberellic acid to stimulate and support spermatogenesis and sex accessory glands resulted an improvement in spermatozoa longevity and sperm enzymatic activity.

Table 7. Milk yield (gm) of NZW rabbit does naturally mated using bucks injected with Gibberellic and Boric acid (Means ± SE).     

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

Table 8. Milk composition and total pre-weaning mortality rates of NZW rabbit does naturally mated using bucks injected with Gibberellic and/ or Boric acid (Means ± SE).     

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

(P ≤ 0.05 or 0.01) different.

Finally, the improvement in litter traits proved that, the Gibberellic and Boric acids treatments are capable to improve the milking ability of the rabbit does which is reflected in her care and ability to suckle her young till weaning.

Regarding mortality rate, Safaa Barakat et al. (2013) reported that, the Gibberellic and Boric acids treatments reduced mortality with about 50% reduction compared with the control. In addition, Gibberellic and Boric acids may be had a role in reduced mortality rate by its role in modify pH of rabbit digestive tract promoting useful bacteria and inhibit the harmful ones (Heindel et al., 1994; Catherine et. al., 1996 and Kamel et al.,2009). Furthermore, the possible antimicrobial activity of the Gibberellic and Boric acids may be accounted for by their growth-promoting effects on bifidobacteria and lactobacilli. These bacteria can reinforce the barrier function of the intestinal mucosa, helping in the prevention of the attachment of pathogenic bacteria, essentially by crowding them out. These bacteria may also produce antimicrobial substances and stimulate antigen specific and nonspecific immune responses (Macfarlane and Cummings, 1999 and Roberfroid, 2000). On the other hand, the milk available per kit may also have a pronounced effect on the mortality of young rabbits (Rommers et al., 2001 and Szendro et. al., 2002).

It is interested to note that, subcutaneously injecting rabbit and does with a   combination of (Gibberellic and Boric acids) showed significantly (P≤0.05 or 0.01) superior parameters than using Gibberellic acid or  Boric acid, alone. 

Conclusively, it could be concluded that: injecting rabbits with a combination of (1 mg Gibberellic acid + 2.5 mg Boric acid) improved semen quality and fertilizing ability of bucks and fertility traits and milk production of does.

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تأثیر حقن حامضى الجبریلیک و البوریک أو معاعلى کفاءة الأرانب التناسلیة

أمل مغاورى هیکل؛ فضیله محمد عیسى؛  صفاء عطایا برکات

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

إن الهدف من هذه الدراسة هو تقییم تأثیر الحقن بحامضى الجبریلیک و البوریک على الکفاءة التناسلیة للأرانب. أستخدم فى هذه الدراسة  244 أرنب نیوزیلندى أبیض (48 ذکر بالغ جنسیاً على عمر 3 أشهر، 196 أنثى على عمر 2,5 شهر).

قسمت الأرانب إلى أربعة مجموعات تجریبیة متجانسة (12 ذکر، و 49 أنثى 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 ملجم حامض الجبریلیک + 2,5 ملجم  من حامض البوریک ، على الترتیب. إشتملت الدراسة على تجربتین أساسیتین، صممت التجربة الأولى لدراسة تأثیر الحقن بحامضى الجبریلیک و البوریک على المقدرة الإخصابیة لذکور أرانب النیوزیلندى البیضاء. أما التجربة الثانیة فقد إهتمت بدراسة تأثیر حقن إناث أرانب النیوزیلندى البیضاء بحامضى الجبریلیک و البوریک على معدلات الخصوبة، وإنتاج وترکیب اللبن، بالإضافة إلى نسبة النفوق خلال مرحلة الرضاعة (قبل الفطام)، وقد تزاوجت الإناث طبیعیاً باستخدام الذکور المعامله بنفس معامله الأمهات.

أوضحت النتائج أن الأوزان المطلقة والنسبیة لکل من (الخصیتین، البربخ، الغدد الجنسیة المساعدة، الغدة النخامیة، محیط کیس الصفن، والدلیل الخصوى، لذکور أرانب النیوزیلندى البیضاء المحقونه بحامضى الجبریلیک و البوریک کانت أفضل معنویاً (عند مستوى 1%) مقارنة بتلک المحقونه بالمحلول الملحى. إنخفض عمر ذکور أرانب النیوزیلندى البیضاء عند أول تلقیحه  معنویاً (عند مستوى 5 %)، بینما وزن الذکور عند أول تلقیحه ونشاطها التزاوجى زاد معنویاً (عند مستوى 5% ), بالإضافة إلى تحسن معنوى (عند مستوى 1%) فى الرغبة الجنسیة والخصائص الطبیعیة للسائل المنوى  نتیجة لحقن الذکور بحامضى الجبریلیک و البوریک.

تحسن معنوی بالنسبة لمعدلات الخصوبة (عند مستوى 5% أو 1%) نتیجة للحقن بحامضى الجبریلیک و البوریک. کانت قیم الناتج من اللبن الکلى، ومعدلات نفوق الصغار خلال مرحلة الرضاعة، من المیلاد وحتى 35 یوم، بالإضافة إلى ترکیب اللبن أفضل معنویاً نتیجة للحقن بحامضى الجبریلیک و البوریک.

التوصیة:  وعموماً، فقد لخصت الدراسة فى مجملها إلى أن حقن الأرانب بحامضى الجبریلیک والبوریک أدى إلى تحسین فى المقدرة الإخصابیة للذکور، وکذلک أدى إلى زیادة فى معدلات خصوبة الإناث.