Trop Anim Health Prod (2013) 45:101–106 DOI 10.1007/s11250-012-0179-0
ORIGINAL RESEARCH
Effect of addition of coconut water (Cocos nucifera) to the freezing media on post-thaw viability of boar sperm María Bottini-Luzardo & Fernando Centurión-Castro & Militza Alfaro-Gamboa & Ricardo Aké-López & José Herrera-Camacho
Accepted: 9 May 2012 / Published online: 15 June 2012 # Springer Science+Business Media B.V. 2012
Abstract The aims of this experiment were to evaluate the addition of coconut water in natura to the freezing media, compare the effect of deionized water vs filtered water of coconut over the post-thaw seminal characteristics, and evaluate the effect of the deionized water and in natura coconut water on the seminal characteristics of boar sperm at different post-thaw times. Thirty-four ejaculates were used divided in three aliquots which received one of the following treatments (T): T1, LEY (bidistilled water, lactose, and egg yolk) and LEYGO (LEY + glycerol and Orvus ET paste); T2, LEYA (coconut deionized water, lactose, and egg yolk)–LEYGOA; and T3, LEYB (in natura coconut water, lactose, and egg yolk)–LEYGOB. Samples of boar semen were frozen according to the Westendorf method, thawed at 38°C, and evaluated at three incubation times (0, 30, and 60 min). Seminal characteristics assessed were motility (Mot), acrosomal integrity (AInt), membrane integrity (MInt), and mitochondrial activity (MAct). T1 showed a higher percentage of viable sperm than T3 (Mot 36.5 vs 5.4 %, AInt 61.8 vs 41.2 %, MInt 50.4 vs 41.3 %, and MAct 56.9 vs 50.5 %). T2 kept a higher percentage of viable sperm at all incubation
M. Bottini-Luzardo : F. Centurión-Castro (*) : M. Alfaro-Gamboa : R. Aké-López Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, km 15.5 Carretera Mérida-Xmatkuil, CP 97100 Mérida, Yucatán, México e-mail:
[email protected] J. Herrera-Camacho Instituto de Investigaciones Agropecuarias y Forestales, Universidad Michoacana de San Nicolás de Hidalgo, Posta Zootécnica, km 9.5 Carretera Morelia-Zinapécuaro, Col. El Trébol, CP 58880 Morelia, Michoacán, México
times. In natura coconut water showed a detrimental effect over the viability of the frozen–thawed boar semen. Deionized coconut water improved the boar semen viability post-thaw, outperforming results of in natura coconut water. Keywords Coconut . Freezing semen . Boars . Post-thawing
Introduction Boar semen is sensitive to changes of temperature, osmolarity, and pH which occur during the cryopreservation process, can cause damage to the membrane and organelles of sperm, and decrease post-thawing survival time and its fertilizing capacity (Grossfel et al. 2008). Due to the advantages of using frozen semen, research has been carried out in order to minimize damages caused by cryopreservation on boar sperm and these have been focused on the modification of protocols and in the addition of substances to the freezing media, which decrease alterations caused to the sperm during this process (Corcuera et al. 2007). In tropical regions, coconut water is a natural and abundant resource, and it has positive effects on the spermatic cell due to its high content of free sugars, antioxidants, and minerals (Vasconcelos et al. 2009). Coconut water has been successfully used for culture and embryo freezing (Martins et al. 2005; Cordeiro et al. 2006) and in canine (Cardoso et al. 2005), goat (Vasconcelos et al. 2009), ovine (Gutiérrez et al. 2006), and boar semen (Kotzias et al. 1999). However, in the case of boar semen, cryopreservation studies have been limited. The objectives of this experiment were to evaluate the effect of the addition of filtered (in natura) and deionized coconut water to the freezing media on the characteristics of boar semen at different post-thawing times.
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Materials and methods The experiment was carried out in a commercial pig farm in the state of Yucatán, México. The climate in the region is Aw0 warm, subhumid with summer rains. Average annual temperature is 27.7°C, relative humidity is 80 %, and the annual mean rainfall is 800 mm (INEGI 2004). Thirty-four ejaculates were used, from six boars from the Pig Improvement Company terminal line 337 (utilizes the variation available in 17 different lines populations worldwide, using breeds as Landrace, duroc, Large white, and other) between 2 and 4 years old, selected as suitable for freezing based on the quality of their semen after freezing. Semen was obtained at 7-day intervals by the gloved hand technique, and only the rich fraction of the sperm was collected (Corcuera et al. 2007). Coconut fruits (Cocos nucifera L. Mexican Pacific tall 2 var.) of approximately 9 years old, obtained from the experimental field from the Center of Scientific Research of Yucatan, were used. Total volume of water was divided into two parts, one part was filtered (filter paper #40) to eliminate particles suspended on it and the other part was subjected to a deionizing process using an electric deionizer (Barnstead, USA) to eliminate excess Ca2+ and using the permanganate method (WPCF 1992); the initial Ca 2+ concentration was 1.82 × 10−2 M and the final concentration was 0 M. Samples were taken (15 ml) separately from the filtered water and from the deionized water to measure pH and osmolarity. The pH was measured with a potentiometer (Corning 7, USA) and osmolarity with an osmometer (Osmette A Automatic Osmometer 5002, USA). When adjustment of pH was required, a 2.9 % sodium citrate solution was added, and the osmolarity was adjusted by dilution with bidistilled water. Both coconut waters (filtered or in natura and the deionized) were stored in a refrigerator at 5°C until used. Three diluents were prepared for the freezing of semen; each one was considered as a treatment (T): the control group or T1, LEY (bidistilled water, lactose, and egg yolk) and LEYGO (LEY + glycerol and Orvus ES paste) (11); the T2, LEYA (deionized coconut water, lactose, and egg yolk)– LEYGOA (LEYA + glycerol + Orvus ET paste); and T3, LEYB (filtered coconut water, lactose, and egg yolk)– LEYGOB (LEYB + glycerol + Orvus ET paste). Immediately after semen collection, a sample was introduced to a water bath at 37°C and a general evaluation to determine seminal volume and spermatic motility was performed. Seminal volume was measured by visual assessment in a graduated test tube, and spermatic motility was evaluated as described by Zemjanis (1974). Samples suitable for freezing were those which presented 80 % of individual motility (Satorre et al. 2007).
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Once the sample was considered suitable for freezing, the Westendorf-modified process was followed for its freezing (Sellos 2001). Straws of 0.54 ml were used to pack the semen by a packing unit (Foruard, France); each straw contained 500 millions of sperm cells. The sealed straws were placed over a steel base, remained there for around 20 min, and exposed to the nitrogen vapors in a polystyrene box at 5 cm away from the surface of the nitrogen; afterwards, they were directly submerged into liquid N2 until analyzed. Evaluation of the seminal viability was carried out 5 days after freezing. Using five straws per treatment, thawing was performed by immersing each straw in a water bath at 38°C for 20 s. The content of each straw was poured in vials with 0.5 ml BTS at 38°C, and the evaluation of the viability was carried out 5 min after thawing (time zero, t0), at 30 min (t30), and at 60 min (t60). Viability of frozen–thawed semen was assessed using the individual motility, evaluated under the microscope at × 40 in a percentage scale of 0–100. If the sample presented 40 % or more of individual motility, it was considered to have a good spermatic motility. Acrosomal integrity (AInt) was evaluated according to Zemjanis (1974). Membrane integrity (MInt) was evaluated using fluorochrome and propidium iodide (Garner et al. 1997), and mitochondrial activity (MAct) was evaluated using the fluorescence technique using rhodamine 123 (Cuello et al. 2001). In both cases, the samples were evaluated at × 1,000 in an epifluorescence microscope (Olympus, Japan). Results were analyzed by ANOVA, the independent variables were the three treatments (T1, T2, and T3), and the response variables were motility (Mot), AInt, MInt, and MAct. Prior to statistical analysis, data were transformed to the arcsine. Means of each treatment were compared by the Duncan test, and P
