|Year : 2017 | Volume
| Issue : 3 | Page : 128-132
Effect of Diospyros kaki enriched extender on cattle bull sperm parameters and conception rate
RI El-Sheshtawy, WS El-Nattat
Animal Reproduction and AI Department., Veterinary Research Division, National Research Centre, Dokki, Egypt
|Date of Web Publication||26-Sep-2017|
R I El-Sheshtawy
Animal Reproduction and Artificial Insemination Department, National Research Centre, Dokki, Giza
Source of Support: None, Conflict of Interest: None
Objective: To explore the effect of Diospyros kaki on cattle spermatozoa during chilling and cryopreservation. Methods: Five milliliter of blended Persimmon (Diospyros kaki) flesh was added to 45 mL TCF to obtain 10% stock solution. Kaki enriched extender (KEE) was prepared by adding to TCF in concentrations 0.0/5.0 mL (control, 0%), 0.5/4.5 mL (1%), 1/4 mL (2%), 1.5/3.5 mL (3%), 2.0/3.0 mL (4%), 2.5/2.5 mL (5%), 3.0/2.0 mL (6%), 3.5/1.5 mL (7%), 4.0/1.0 mL (8%), 4.5/0.5 mL (9%) and 5.0/0.0 mL (10%) to obtain a final volume 5 mL in each tube. Whole egg yolk was added to each tube to obtain KEE with 20% egg yolk (KEEY), all tubes were centrifuged to get rid of debris. Semen was added to the supernatants in other tubes. Extended semen was subjected to evaluation [motility, alive sperm and intact sperm membrane (HOST) %] in both chilled and cryopreserved semen. Conception rate was carried out. Results: Sperm motility was significantly (P<0.000 1) kept high after 11 d of chilling with the concentration 1%, 2%, 3%, 4%, 5% as compared to the control (41.67±1.67, 41.67±1.67, 40.00±0.00, 41.67±1.67 and 41.67±1.67, respectively) and also non-significantly kept high at the other concentrations up to 9 d of chilling. Addition of KEE had significantly (P<0.003 3) improved post thawing sperm motility % with the concentrations 1, 2, 3, 4, 5 and 6% as compared to the control (51.67±5.27, 55.00±3.16, 48.33±1.05, 45.00±3.96, 57.00±2.50, 55.00±5.00 and 43.33±5.11 respectively).While the other concentrations exhibit no effect. Addition of KEE maintained alive sperm%, abnormalities% and % of intact spermatozoa membranes (HOST%) as good as the control with all concentrations of kaki used in our study. The conception rate upon using frozen semen in insemination showed higher conception rate in concentrations of 2%, 4% and 6 % KEE in cattle. Conclusion: It could be concluded that some concentrations of Diospyros kaki improved bull semen quality post-chilling and post-freezing.
Keywords: Cattle, Semen, Preservation, Diospyros kaki
|How to cite this article:|
El-Sheshtawy R I, El-Nattat W S. Effect of Diospyros kaki enriched extender on cattle bull sperm parameters and conception rate. Asian Pac J Reprod 2017;6:128-32
|How to cite this URL:|
El-Sheshtawy R I, El-Nattat W S. Effect of Diospyros kaki enriched extender on cattle bull sperm parameters and conception rate. Asian Pac J Reprod [serial online] 2017 [cited 2021 May 6];6:128-32. Available from: http://www.apjr.net/text.asp?2017/6/3/128/215616
| 1. Introduction|| |
Spermatozoa are the endpoint of male spermatogenesis and have particular anatomic and metabolic features. Nowadays, Sperm cryopreservation and storage are of a great demand for conserving the supergenetic origins of the males, the development of artificial reproductive technologies such as artificial insemination (AI) and in vitro fertilization (IVF) are of a great interest. AI with frozen semen is essential in breeding and selection schedules contributing to increase production of domestic species. Nowadays, semen cryopreservation has many biotechnological applications. It can be used to solve infertility problems, life threatening diseases, preservation of semen and DNA from endangered species and conservation of biodiversity. The interaction of several factors (cooling rate, storage temperature, chemical ingredients of the extender, cryoprotectant concentration, over accumulation of oxygen free radicals, seminal plasma composition and hygienic control) are the key that affect the life-span of spermatozoa. Cryopreservation of bovine semen often induce an additional source for reactive oxygen species (ROS) attack on sperm due to decreased activities of antioxidant enzymes and the sperm membrane become more susceptible to lipid peroxidation which affect the membrane permeability. Natural antioxidants exert a protective effect preserving the metabolic activity and cellular viability of cryopreserved bovine spermatozoa. Nowadays, the use of herbal natural product has gained interest worldwide. Many of the herbs have been developed into herbal supplement which are claimed to assist in healthy life style. Among these herbs, is Persimmon (Diospyros kaki) fruit possessing high nutritive value and contains many biologically active substances including antioxidants, dietary fiber, triterpenoids and minerals,,,,,,. Persimmons revealed considerable health and medicinal benefits, which are considered to be related to the various hydrophilic and lipophilic antioxidants including phenolic compounds, vitamin C and carotenoids, contained in the fruit. Persimmon fruits contain abundance of nutrients and phytochemicals such as carbohydrates, organic acid, vitamins, tannins, polyphenols, dietary fiber, triterpenoids, and carotenoids, which contribute significantly to their taste, colour, nutritive and medicinal values,,,,,,,.
No available literatures were found for interpreting the benefit for using those materials in preserving extended cattle and buffalo semen.
| 2. Materials and methods|| |
2.1. Preparation of semen extender
Kaki enriched extender (KEE): 5 mL of blended Persimmon (Diospyros kaki) flesh was added to 45 mL TCF to obtain 10% stock solution. KEE was prepared by adding to TCF in concentrations 0.0/5.0 mL (control, 0%), 0.5/4.5 mL (1%), 1/4 mL (2%), 1.5/3.5 mL (3%), 2.0/3.0 mL (4%), 2.5/2.5 mL (5%), 3.0/2.0 mL (6%), 3.5/1.5 mL (7%), 4.0/1.0 mL (8%), 4.5/0.5 mL (9%) and 5.0/0.0 mL (10%) to obtain a final volume 5 mL in each tube. Whole egg yolk was added to each tube to obtain KEE with 20% egg yolk (KEEY), all tubes were centrifuged to get rid of debris. Supernatants were transferred to other tubes and then stored at -20 °C till used.
A preliminary study on the use of 10%-50% had given bad results so, the former design was introduced as the persimmon contains high amount of astringent tannin which may be the cause of those poor results.
2.2. Semen collection and initial evaluation
Three mature cattle bulls maintained at Semen Freezing Center, General Organization for Veterinary Services Ministry of Agriculture, Abbasia, Egypt, were used as semen donors. Ejaculates were collected using a bovine adapted artificial vagina at weekly intervals for 18 wk. Semen samples were initially evaluated for subjective sperm motility and sperm concentration. Ejaculates fulfilling minimum standard of sperm motility (70%) and sperm morphology pooled in order to have sufficient semen for a replicate and to eliminate the bull effect. The semen was given a holding time for 10 min at 37 °C in the water bath before dilution.
2.3. Semen processing
Semen samples were diluted with TCF extender and used as control and other aliquots of pooled semen samples were diluted with TCF extenders containing the different concentrations of kaki in order to provide concentration of 60 million sperm/mL. Extended semen was slowly cooled (approximately for 2 h) to 5 °C and equilibrated for 2 h. Semen was packed into 0.25 mL polyvinyl French straws. After equilibrium periods, the straws were horizontally placed on a rack and frozen in vapor 4 cm above liquid nitrogen for 10 min and were then dipped in liquid nitrogen. A fraction of extended semen from control and each concentration of the additives were kept at 5 C for 7-10 d (chilling) and sperm motility was evaluated daily.
2.4. Assessment of semen quality parameters
The assessment was undertaken on after freeze thawing of bull spermatozoa. Also, sperm motility was evaluated for raw semen, 2 h after cooling and chilled semen daily up to 7-10 d. Frozen straws were thawed at 37 °C for 1 min. The parameters studied were subjective semen characteristics (motility, alive, abnormality and hypoosmotic swelling test (HOST) %).
2.5. Conception rate
Conception rate no. of cows (n=145) were inseminated with KEEY bull semen. Another no. of cows was inseminated with bull semen diluted with TCFY (control group). Pregnancy was confirmed by rectal palpation 2 mo later after insemination. The inseminated cows were used via the cooperation in Beni-Suef Governorate. CR was calculated according to the equation:
2.6. Statistical analysis
Statistical analysis data were analyzed using the SPSS (2005) computerized program v. 14.0 to calculate the analysis of variance (ANOVA) for the different parameters between control and additives replications. Significant difference between means was calculated using Duncan test at P<0.05.
| 3. Results|| |
3.1. Effect of Diospyros kaki enriched extender on cattle bull sperm motility during chilling
Sperm motility was significantly (P<0.000 1) kept high after 11 d of chilling with the concentration 1%, 2%, 3%, 4%, 5% as compared to the control (41.67±1.67, 41.67±1.67, 40.00±0.00, 41.67±1.67 and 41.67±1.67, respectively) and also non- significantly kept high at the other concentrations up to 9 d of chilling [Table 1].
|Table 1: Effect of Diospyros kaki enriched extender on cattle bull sperm motility during chilling.|
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3.2. Effect of Diospyros kaki enriched extender on post thawing cattle bull sperm characteristics
Addition of KEE had significantly (P<0.003 3) improved post thawing sperm motility% with the concentrations 1%, 2%, 3%, 4%, 5% and 6% as compared to the control (51.67±5.27, 55.00±3.16, 48.33±1.05, 45.00±3.96, 57.00±2.50, 55.00±5.00 and 43.33±5.11 respectively) while the other concentrations exhibit no effect. Addition of KEE maintained alive sperm%, abnormalities% and % of intact spermatozoa membranes (HOST %) as good as the control with all concentrations of kaki used in our study [Table 2].
|Table 2: Effect of Diospyros kaki enriched extender on post thawing cattle bull sperm characteristics.|
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The conception rate [Table 3] upon using frozen semen in insemination showed higher conception rate in concentrations of 2%, 4% and 6 % KEE in cattle.
|Table 3: Effect of Diospyros kaki enriched extender on a field conception rate test in cattle.|
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| 4. Discussion|| |
Cryopreservation of sperm is of a great demand. According to Gadea et al., Uysal and Bucak and Bucak et al., minimizing the physical and chemical stresses of cooling, freezing and thawing of sperm cells and consequently improving viability and subsequent fertilizing capacity is achieved by including cryoprotectants in the semen extender. Cryopreservation causes wide-ranging physical, chemical and mechanical injures to sperm membranes of all mammalian species, which are attributed to temperature changes, alterations in the transition from the lipid phase, production of reactive oxygen species (ROS) and osmotic stress,. Moreover, the over accumulation of ROS causes a state of oxidative stress that involves structural damage of sperm membranes, fall of intracellular ATP levels causing decreasing in the viability and motility of cryopreserved sperm,. To decrease the harmful effects of ROS, seminal plasma possesses powerful source of ROS scavengers which offer protection for equine sperm, including enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and small molecular antioxidants such as ascorbic acid and α-tocopherol,. The herbal remedies contain antioxidants to counteract the deleterious action of reactive oxygen species (ROS).
The results of the present trials revealed that sperm motility of cattle semen was kept high up to 11 d of chilling in KEEY with the concentrations 1%, 2%, 3%, 4% and 5% as compared to the control. This means that semen with these concentrations could be used in AI up to 11 d of chilling. Also, with the other concentrations of KEEY semen could be used in AI up to 9 d of chilling. On the other hand, KEEY significantly improved post thawing sperm motility with the concentrations 1%, 2%, 3%, 4%, 5% and 6% and also maintained alive sperm%, abnormalities and % of intact spermatozoa membranes. Conversely, KEEY had improved the post thawing sperm motility with all concentrations (1%-9%) except the 10%. All the used concentrations (1%-10%) had maintained alive %, Abnormalities% and % of intact spermatozoa membranes (HOST %). Higher conception rate in cattle with concentrations 2% and 4% kaki (62.50% and 66.67% respectively) coincide with the good post-thawing sperm parameters with these concentrations (sperm motility 55.00±3.16 and 45.00±3.96 respectively, Host 88.67±1.67 and 86.67±2.33 respectively, alive 84.67±1.20 and 80.33±0.33 respectively, abnormalities 17.33±0.58 and 8.67±1.76, respectively). Higher conception rate with the 6% kaki concentration (60.00%) come in accordance with the higher post thawing sperm parameters with this concentration (sperm motility 55.00±5.00, Host 83.33±2.85, alive 82.67±0.33 and abnormalities 16.33±3.84).
Cryopreservation of bovine semen often exert an additional source of reactive oxygen (ROS) attack and lipid peroxidation which affects the membrane permeability. The increased ROS level is due to the decreased activities of antioxidant enzymes. Natural antioxidants protect and preserve the metabolic activity and cellular viability of cryopreserved bovine spermatozoa. Diospyros kaki is rich in strong antioxidants as represented by high contents of carotenoids,, flavonoids and polyphenols,,, organic acids and vitamins,, carbohydrates, and minerals. Olayemi et al. and Aljady et al. recorded that phenolic acids and flavonoids have strong antioxidant capacity and improved semen quality of preserved semen. Flavonoids are excellent scavengers of free radicals and the number of hydroxyl group on the phenyl ring enhances the antioxidant capacity of polyphenolic molecule,. Kaki is rich in flavonoids which have strong antioxidants activity and suppressing the production of malondialdehyde and nitric oxide and inhibit apoptosis. El-Sheshtawy et al., conclude that pollen grains and honey improved preservability of bull semen due to its high contents of antioxidants flavonoids, vitamins and organic acids. It could be concluded that natural additives to semen extenders (moringa, kaki and silymarin) improved semen preservability in both chilled and frozen semen.
Conflict of interest statement
The authors hereby certify that they have no conflict of interest.
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[Table 1], [Table 2], [Table 3]