• Users Online: 385
  • Print this page
  • Email this page

Table of Contents
Year : 2017  |  Volume : 6  |  Issue : 6  |  Page : 279-282

Cryopreservation of cattle semen using coconut water extender with different glycerol concentrations

Animal Reproduction and Al Department, Veterinary Division, National Research Centre, Dokki, Giza, Egypt

Date of Submission22-Aug-2017
Date of Decision01-Sep-2017
Date of Acceptance15-Sep-2017
Date of Web Publication01-Nov-2017

Correspondence Address:
Reda Ibrahim El-Sheshtawy
Animal Reproduction and Artificial Insemination Department, Veterinary Research Division, 13 National Research Centre, Dokki, Giza
Walid Said El-Nattat
Animal Reproduction and Artificial Insemination Department, Veterinary Research Division, National Research Centre, Dokki
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2305-0500.217343

Rights and Permissions

Objective: To investigate the effect of coconut water with a lone concentration and different concentrations of glycerol on chilled and cryopreserved cattle semen characteristics. Methods: Semen was collected from five mature cattle bulls, at weekly intervals for 5 weeks. The ejaculates were pooled and evaluated for dilution processing. Tris citrate egg yolk fructose was used as control treatment for semen, while 50% (V/V) coconut water, 25% (V/V) bi-distilled water and 25% (V/V, 5% anhydrous monosodium citrate) to 20 mL egg yolk and three different concentrations of glycerol (4%, 6% and 8%) were used as coconut water (CW)- glycerol-yolk extenders (CWCG-4, CWCG-6 and CWCG-8). Extended semen was cooled and cryopreserved. Sperm motility%, sperm membrane integrity%, normal acrosome%, live sperm% and total sperm abnormalities% were recorded after equilibrium and after freeze-thawing. Results: The addition of 4% glycerol to coconut water enriched media (CWCG-4) revealed the most effective addition of glycerol on all parameters after equilibrium and after freeze-thawing. Conclusions: Coconut water enriched media with 4% glycerol addition is safe to be used as an extender in bull semen preservation because it is a sterile liquid. So, it can be used without addition of antibiotics to the extender, as antibiotics have to some extent hazardous effect on spermatozoa.

Keywords: Semen, Cattle, Glycerol, Coconut water, Freeze thawing

How to cite this article:
El-Sheshtawy RI, El-Nattat WS, Ali GD. Cryopreservation of cattle semen using coconut water extender with different glycerol concentrations. Asian Pac J Reprod 2017;6:279-82

How to cite this URL:
El-Sheshtawy RI, El-Nattat WS, Ali GD. Cryopreservation of cattle semen using coconut water extender with different glycerol concentrations. Asian Pac J Reprod [serial online] 2017 [cited 2023 Mar 21];6:279-82. Available from: https://www.apjr.net/text.asp?2017/6/6/279/217343

  1. Introduction Top

Natural extracts from vegetables and fruits and their seeds maintain life due to their high contents of medicinal compounds essential for health[1],[2]. These natural extracts and infusions are used in semen extenders for preserving animal sperms[3]. This cryopreserving property is mainly related to their strong antioxidant capacity, thus protecting spermatozoa from oxidative damage during cryopreservation process[4],[5]. Coconut water is characterised by its high contents of antioxidants as expressed by the phytohormones[6], sugar, vitamins, electrolytes and amino acids[7]. The suitability of coconut water-based diluents for processing buffalo semen was shown by Vale et al[8],[9] and El-Nattat et al[10]. Coconut water is capable of in vitro sperm capacitation in swine[11] and as a maturation and culture medium for bovine[12], caprine[13] and ovine[14] oocytes and embryos.

The main objective of this study was to evaluate coconut water with different concentrations of glycerol (4%, 6% and 8%) as a semen extender for preserving cattle semen as reflected on sperm motility, sperm membrane integrity, viable and abnormal sperm percent and acrosome integrity of cooled and frozen semen.

  2. Materials and methods Top

2.1. Semen collection and initial evaluation

Semen was collected from five mature cattle-bulls, reared at Buffalo Semen Freezing Centre, General Organization for Veterinary Services, Ministry of Agriculture, Abbasia, Egypt, using an artificial vagina at weekly intervals for 5 weeks. The semen was transferred to the laboratory within few minutes. Semen samples were examined for volume, percentage of motile sperm, general sperm morphology and sperm cell concentration. Semen samples with more than 70% motility and 80% morphologically normal spermatozoa and 1×109/mL sperm concentration were used for dilution and processing. The ejaculates were pooled in order to have sufficient semen for replicates and to eliminate the bull effect. The semen was given a holding time for 10 min in a water bath at 35-37 °C before dilution.

2.2. Semen processing

Tris-citric acid-fructose-egg yolks (TCFY) were used as a control extender[15]. Coconut water obtained from a tender, green, healthy and undamaged coconut fruit coconut water (CW) was filtered two times. CW extender with 20% egg yolk (v/v) and one of three different glycerol concentrations (4%, 6% and 8% V/V) was prepared and used according to Vale et al[8] and Cardoso et al[16]. All extenders contained 1 000 IU penicillin and 1 mg streptomycin per milliliter.

Pooled semen samples were split into four equal fractions and diluted at 37 °C in a single step with the four experimental extenders (TCFY and CW containing 4%, 6% and 8% glycerol) to a final concentration of 60×106 spermatozoa/mL. Extended semen fractions were exposed to freezing process according to Ahmad et al[17].

2.3. Semen quality assessment

Sperm motility%, sperm membrane integrity%, normal acrosome%, live sperm% and total sperm abnormalities% were assessed after cooling and freeze-thawing (frozen straws were thawed at 37 °C for one minute).

2.3.1. Sperm motility

Sperm motility was assessed according to Graham et al[18].

2.3.2. Sperm membrane integrity

Plasma membrane integrity of buffalo bull spermatozoa was assessed using the hypo-osmotic swelling test (HOST) as described by Jeyendran et al[19] and Ahmad et al[17].

2.3.3. Percentage of live sperm and normal acrosome

The dual staining procedure with trypan blue-giemsa stain was performed as described by Kovacs and Foote[20].

2.3.4. Sperm morphology

Abnormal sperm percent was examined in eosin nigrosine stained semen smears[21].

2.4. Statistical analysis

The results expressed as mean± standard errors of the mean. To compare coconut extender with different glycerol concentrations to the control Tris, the data were analyzed by analysis of variance using the ANOVA procedure of SAS program v. 9.2[22]. Differences between means were compared with Waller-Duncan multiple range at Kratio=100.

  3. Results Top

Data output revealed that the cooled semen membrane integrity and acrosome integrity percentages were the only parameters to be affected by the variation of glycerol addition and CW [Table 1]. The addition of 4% glycerol to CW enriched media revealed the most effective addition of glycerol on all parameters. The motility and livability % were apparently increased while the abnormality % was decreased when compared to other treatments. Only HOST and acrosome were significantly (P<0.001) affected.
Table 1: Coconut enriched media with different concentrations of glycerol effect on cooled cattle semen (Mean± SE) (%).

Click here to view

In the same consent, after freeze-thaw, the addition of variant glycerol concentrations to CW enriched media had significantly affected all parameters (percentage of motility, livability, abnormality, HOST and acrosome) [Table 2]. The CW with 4% glycerol revealed highly significant percentage for motility, livability, HOST and acrosome integrity (48.78±1.57, 81.67±1.67, 76.25±0.59 and 76.67±1.67, respectively) compared to the other treatments. While the abnormality percentage was significantly the lowest (16.67±0.88) compared to the other treatments.
Table 2: Coconut enriched media with different concentrations of glycerol effect on frozen cattle semen (Mean± SE) (%).

Click here to view

  4. Discussion Top

Recently, scientists are interested in the potential health benefits of phytochemicals and the synergistic effects of their multiple compounds compared to the single purified active fractions[4]. Semen cryopreservation leads to biochemical and functional damage to spermatozoa, thus reducing its motility and viability[18], but it is important to preserve the valuable genetic constitution of our local breeds of cattle bulls. Cryodamage induced by freezing and thawing can be decreased by adding lipoproteins, or using the suitable cryoprotectant in the semen extender[23]. Semen freezing is associated with over production of reactive oxygen species and reduction in the antioxidant capacity as manifested by a decrease in intracellular GSH content that induce damage in spermatozoal membrane[24],[25],[26]. Seminal plasma has limited antioxidant capacity, so, the use of an extender having strong antioxidant effect is recommended to maintain the viability and fertilizing capacity of frozen spermatozoa[26]. Motility is the most important parameter used for semen evaluation, both before and after preservation[27]. Concannon and Battista[28] suggest that at least 40%-50% sperm motility is necessary for success in artificial insemination. However, Linde-Forsberg and Forsberg[29] postulated that 20%-30% sperm motility is necessary for pregnancy. In our study, CW extender with glycerol 4%, 6% and 8% improved sperm membrane integrity (HOST%) of cooled semen and the superior result was obtained in CW with 4% glycerol. In frozen semen CW extender with glycerol 4% was able to preserve sperm quality as manifested by higher motility, alive, HOST, intact acrosome % and lowered abnormalities as compared to Tris control extender. Silva et al[30],[31] concluded that CW containing glycerol is recommended to achieve satisfactory post freezing quality of boar semen. Cardoso et al[32] recorded that cryopreservation of canine semen using CW extender resulted in a higher quality of canine sperm. Canine chilled extended semen in CW provided good pregnancy and whelping rates with a higher percentage of female births from French bitches[33]. The inclusion of coconut milk at 15% and 20% in tris-extenders markedly improved sperm viability parameters of goat buck semen[34]. Non-permeable coconut ingredients and permeable glycerol cryoprotectants induce a synergistic beneficial effect on spermatozoa during freezing. The improved results in our study is attributed to the inclusion of CW with high contents of phytohormones[6], sugars[7], vitamins[35], electrolytes[36] and amino acids[37]. All these ingredients in CW have strong antioxidant activities that protect sperm against oxidative damage. CW enriched media with 4% glycerol addition (CWCG-4) is safe to be used as an extender in bull semen preservation because it is a sterile liquid, so, it can be used without addition of antibiotics to the extender, as antibodies have to some extent hazardous effect on spermatozoa.

Conflict of interest statement

We declare that we have no conflict of interest.

  References Top

Aviram M, Dornfield L, Rosenblatt M, Volkova N, Kaplan M, Coleman R. Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice. Am J Clin Nutr 2000; 71: 1062-1076.  Back to cited text no. 1
Aviram M, Rosenblatt M, Gaitani D, Nitecki S, Hoffman A, Dornfield L. Pomegranate juice consumption for 3 years by patients with carotid artery stenosis (CAS) reduces common carotid intima-media thickness (IMT), blood pressure and LDL oxidation. Clin Nutr 2004; 23: 423-433.  Back to cited text no. 2
Sansone G, Nastri MJF, Fabbrocini A. Storage of buffalo (Bubalus bubalis) semen. Anim Reprod Sci 2000; 62: 55-76.  Back to cited text no. 3
Seeram NP, Adams LS, Henning SM, Niu Y, Zhang Y, Muraleedharan G, et al. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, Ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. J Nutr Biochem 2005; 16: 360-367.  Back to cited text no. 4
Tezcan F, Ozguven MG, Diken T, Ozcelik B, Erim FB. Antioxidant activity and total phenolic, organic acid and sugar content in commercial pomegranate juices. Food Chem 2009; 115: 873-877.  Back to cited text no. 5
Nunes JF, Sales FGM. El agua de coco (Cocus nucifera) in natura integral y adicionada con citoquinina, comodiluidor de semen caprino. Rev Ciencia Anim 1993; 3(3): 273- 281.  Back to cited text no. 6
Yong JWH, Ge L, Fei Ng Y, Tan SN. The chemical composition and biological properties of coconut (Cocos nucifera L.) water. Molecules 2009; 14: 5144-5164.  Back to cited text no. 7
Vale WG, Silva AOA, Sousa JS, Ribeiro HFL, Souza HEM, Nahum BS, et al. Preliminary report on the use of coconut water (Cocos nucifera) as a diluter of buffalo semen. Proceeding of the 5th World Buffalo Congress. Royal Palace, Caserta-Italy; 1997, p. 836-839.  Back to cited text no. 8
Vale WG, Nahum BS, Silva AOA, Sousa JS, Souza HEM, Ohashi OM, et al. Inseminação artificial em búfalos com sêmen congelado em diluentea base de água de coco (Cocos nucifera). Rev Bras Reprod Anim 1999; 23: 354-356.  Back to cited text no. 9
El-Nattat WS, El-Sisy GA, El-Sheshtawy RI. Cryopreservation of buffalo semen using coconut water extender with egg yolk and different glycerol concentrations. Egypt J Basic Appl Physiol 2009; 8: 139-146.  Back to cited text no. 10
Nascimento AB, Oliveira VP, Marques MG, Toniolli R, Gerger RPC, Coutinho ARS, et al. Dehydrated coconut water for in vitro sperm capacitation in swine. Reprod Fert Dev 2005; 17: 275 (Abstr. 250).  Back to cited text no. 11
Lucci CM, Kacinskis MA, Rumpf R, Báo SN. Effects of lowered temperatures and media on shorter preservation of zebu (Bos indicus) preantral ovarian follicles. Theriogenology 2004; 61: 461-472.  Back to cited text no. 12
Silva JRV, Van Den Hurk R, Costa SHF, Andrade ER, Nunes APA, Ferreira FVA, et al. Survival and growth of caprine primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water. Anim Reprod Sci 2004; 81: 273-286.  Back to cited text no. 13
Andrade ER, Amorim CA, Matos MHT, Rodrigues APR, Silva JRV, Man D, et al. Evaluation of saline and coconut water solutions in preservation of ovine preantral follicles in situ. Small Ruminant Res 2002; 43: 235-243.  Back to cited text no. 14
Foote RH. Fertility of bull semen at high extension rates in tris buffered extenders. J Dairy Sci 1970; 53: 1475-1477.  Back to cited text no. 15
Cardoso RCS, Silva AR. Silva LDM. Comparison of two dilution rates on canine semen quality after cryopreservation in a coconut water extender. Anim Reprod Sci 2006; 92: 384-391.  Back to cited text no. 16
Ahmad Z, Anzar M, Shahab M, Ahmad N, Andrabi SMH. Sephadex and sephadex-ion exchange filtration improves the quality and freezability of low-grade buffalo ejaculates. Theriogenology 2003; 59: 1189-1202.  Back to cited text no. 17
Graham EF, Schmehl MKL, Maki-Laurila M. Some physical and chemical methods of evaluating semen. In: Proeding 3rd NAAB Technology Conference Artificial Insemination Reproduction 12-14 April Milwaukee, WI. National Association of Animal Breeders, Columbia, MO; 1970, p. 44- 48.  Back to cited text no. 18
Jeyendran RS, Van der Van HH, Perez-Pelaez M, Crabo BG, Zaneveld LJD. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fert 1984; 70: 219-228.  Back to cited text no. 19
Kovacs A, Foote RH. Viability and acrosome staining of bull, boar and rabbit spermatozoa. Biotechnol. Histochem 1992; 67: 119-124.  Back to cited text no. 20
Gormier N, Sirard MA, Baile JL. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J Androl 1997; 18: 461-468.  Back to cited text no. 21
Statistical analysis system. User's guide v. 9.2. Cary N.C., USA: Inst. Inc. 2008.  Back to cited text no. 22
Agarwal A, Prahakaran SA, Said TM. Prevention of oxidative stress injury to sperm. J Androl 2005; 26: 653-660.  Back to cited text no. 23
Ball BA, Medina V, Gravance CG, Baumber I. Effect of antioxidant on preservation of motility, viability and acrosomal integrity of equine spermatozoa during storage at 5 °C. Theriogenology 2001; 56: 577-569.  Back to cited text no. 24
Bilodeau JF, Blanchette S, Gagnon IC, Sirard MA. Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen. Theriogenology 2001; 56: 275-286.  Back to cited text no. 25
Gadea J, Selles E, Marco MA, Coy P, Matas C, Romar R, et al. Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders. Theriogenology 2004; 62: 690-701.  Back to cited text no. 26
Rota A. Studies on preservation, capacitation and fertility of dog spermatozoa. Thesis. Uppsala, Sweden: Swedish University of Agricultural Sciences; 1998.  Back to cited text no. 27
Concannon PW, Battista M. Canine semen freezing and artificial insemination. In: Kirk RW, editor. Current veterinary therapy. Philadelphia: W.B. Saunders Company; 1989.  Back to cited text no. 28
Linde-Forsberg C, Forsberg M. Fertility in dogs in relation to semen quality and the time and site of insemination with fresh or frozen semen. J Reprod Fert 1989; 39: 299-310.  Back to cited text no. 29
Silva MA, Peixoto GCX, Lima GL, Bezerra JAB, Campos LB, Paiva ALC, et al. Cryopreservation of collared peccaries (Tayassu tajacu) semen using a powdered coconut water (ACP-116c) based extender plus various concentrations of egg yolk and glycerol. Theriogenology 2012; 78: 605-611.  Back to cited text no. 30
Silva CG, Cunha ER, Blume GR, Malaquias JV, Bào SN, Martins CF. Cryopreservation of boar sperm comparing different cryoprotectants associated in media based on powdered coconut water, lactose and trehalose. Cryobiology 2015; 70: 90-94.  Back to cited text no. 31
Cardoso RCS, Silva AR, da Silva LDM. Comparison of two dilution rates on canine semen quality after cryopreservation in a coconut water extender. Anim Reprod Sci 2006; 92: 384-391.  Back to cited text no. 32
Uchoa DC, Pereira da Silva TF, Cardoso J de FS, Filho ACM, Jucá RP, Silva AR, et al. Favoring the birth of female puppies after artificial insemination using chilled semen diluted with powdered coconut water (ACP-106c). Theriogenology 2012; 77: 1959-1963.  Back to cited text no. 33
Daramola JO, Adekunle EO, Iyasere OS, Oke OE, Sorongbe TA, Iyanda OA, et al. Effects of coconut milk alone or supplementation with pyridoxine in tris-extenders on viability of buck spermatozoa during vitrification. Small Ruminant Res 2016; 136: 208-213.  Back to cited text no. 34
Leong LP, Shui G. An investigation of an antioxidant capacity of fruits in Singapore markets. Food Chem 2002; 76: 69-75.  Back to cited text no. 35
USDA. USDA National Nutrient Database. Coconut meat, raw, (Cocus nucifera), fresh, nutrition value per 100 g. 2015. [Online] Available from: http://www.nutrition-and-you.com/coconut.html.  Back to cited text no. 36
Toniolli R, Bussiere J, Courot M, Magistrini M, Combamous Y. Effect of indole-3-acetic acid (plant Auxins) on the preservation at 15 С of boar semen for artificial insemination. Reprod Nutr Dev 1996; 36: 503-511.  Back to cited text no. 37


  [Table 1], [Table 2]

This article has been cited by
1 Effect of graphene oxide as cryoprotectant on post-thaw sperm functional and kinetic parameters of cross bred (HF X Sahiwal) and Murrah buffalo ( ) bulls
Prahlad Singh, Munish Kumar Bedi, Sumit Singhal, Ashwani Kumar Singh, Ajeet Kumar, Mrigank Honparkhe
Cryobiology. 2022;
[Pubmed] | [DOI]
2 Potential of watermelon (Citrullus lanatus) to maintain oxidative stability of rooster semen for artificial insemination
Olatunji Abubakar Jimoh,Micheal Olawale Akinola,Bolaji Fatai Oyeyemi,Wahab Adekunle Oyeyemi,Simeon Olugbenga Ayodele,Idowu Samuel Omoniyi,Hafsat Ololade Okin-Aminu
Journal of Animal Science and Technology. 2021; 63(1): 46
[Pubmed] | [DOI]
3 Comparison of conventional and automated freezing methods on PB2 rooster semen cryopreserved with glycerol and dimethylsulfoxide tris coconut-water extender
Adedeji Suleimon Balogun,Raman Narang,Ranjna S. Cheema,Ajeet Kumar,Narinder Singh,Malik Vijaysingh
Bulletin of the National Research Centre. 2020; 44(1)
[Pubmed] | [DOI]
4 Potential of coconut water to enhance fresh semen quality and fertility in rabbits
O. A. Jimoh
Tropical Animal Health and Production. 2019;
[Pubmed] | [DOI]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  2. Materials and...
  In this article
1. Introduction
3. Results
4. Discussion
Article Tables

 Article Access Statistics
    PDF Downloaded640    
    Comments [Add]    
    Cited by others 4    

Recommend this journal