Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell extender

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1 Available online at Theriogenology 74 (2010) Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell extender S. Akhter*, M.S. Ansari, B.A. Rakha, S.M.H. Andrabi, S. Iqbal, N. Ullah Animal Physiology Laboratory, Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi-46300, Pakistan Received 6 June 2009; received in revised form 21 April 2010; accepted 22 April 2010 Abstract This study was designed to compare commercially available extender Bioxcell with tris-citric egg extender for post thaw quality and in vivo fertility of buffalo semen. For comparison of post thaw semen quality: semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina (at 42 C) for three weeks (replicates). Qualifying ejaculates having motility 60% from each buffalo bull were divided in two aliquots and diluted (at 37 C having spermatozoa/ml) in tris-citric egg or Bioxcell extender. Diluted semen was cooled to 4 C in 2 hours, equilibrated for 4 hours and filled in 0.5 ml straws. Semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. Straws were then plunged and stored in liquid nitrogen ( 196 C). After 24 hours of storage, semen straws were thawed at 37 C for 30 seconds to assess sperm motility, viability, plasma membrane integrity, normal apical ridge, and abnormalities (head, mid piece, and tail). For comparison of in vivo fertility: semen from two buffalo bulls of known fertility was cryopreserved in tris-citric egg and Bioxcell as described earlier, and used for inseminations under field conditions. Post-thaw percentage of sperm motility ( , ), viability ( , ) plasma membrane integrity ( , ) and normal apical ridge ( , ) did not differ (P 0.05) in tris-citric egg and Bioxcell extender, respectively. Similarly, sperm abnormalities of head ( , ), mid piece ( , ) and tail ( , ) remained similar (P 0.05) in tris-citric egg and Bioxcell extender, respectively. In vivo fertility rates of buffalo semen cryopreserved in tris-citric egg and Bioxcell also remained similar (44% vs. 47%). It is concluded that commercially available Bioxcell may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg extender Elsevier Inc. All rights reserved. Keywords: Bioxcell ; Tris-citric-egg ; Buffalo semen; Cryopreservation 1. Introduction Tris-citric egg extender has been widely recommended for routine use to cryopreserve buffalo bull semen [1]. However, the freezability and fertility rates with cryopreserved buffalo semen are lower using triscitric egg extender compared to fertility rates using cryopreserved semen in cattle [1,2]. It requires * Corresponding author. Tel.: address: sashraf1993@gmail.com (S. Akhter). continuous experimentation to identify the extender for cryopreservation of semen which can produce acceptable fertility rates in buffalo. Commercially available soya lecithin based extenders AndroMed [3 5], Biociphos plus [5 10] and Bioxcell have been used for the cryopreservation of bovine, caprine and ovine semen. Cryopreservation of caprine [11,12] and bovine [13 15] semen in soya lecithin based extender Bioxcell maintained the semen quality (motility, plasma membrane integrity and acrosmal integrity) and produced acceptable fertility X/$ see front matter 2010 Elsevier Inc. All rights reserved. doi: /j.theriogenology

2 952 S. Akhter et al. / Theriogenology 74 (2010) rates [12]. Based on these results, it was decided to investigate Bioxcell as an extender for the cryopreservation of buffalo semen. It was hypothesized that cryopreservation of buffalo semen in Bioxcell might result in an improved post thaw semen quality and in vivo fertility. The present study was designed to compare the post thaw sperm motility, viability, plasma membrane integrity, acrosome integrity, morphology, and in vivo fertility of buffalo semen cryopreserved in commercially available extender Bioxcell and tris-citric egg extender. 2. Materials and Methods 2.1. Preparation of extenders Tris-citric egg extender was prepared by using 1.56 g citric acid (Fisher Scientific, UK) and 3.0 g tris (hydroxymethyl)-aminomethane (Research Organics, USA), fructose (Scharlau, Spain) 0.2% wt/vol, glycerol 7.0 ml (Merck, Germany), and egg 20% in 74 ml distilled water. [16]. Antibiotics namely gentamycin sulphate (500 g/ml; Reckitt Benckiser, Pakistan), tylosin tartrate (100 g/ml; VMD, Belgium), lincomycin hydrochloride (300 g/ml; Pharmacia & Upjohn, Belgium) and spectinomycin hydrochloride (600 g/ml; Pharmacia & Upjohn, Belgium) were added to tris-citric egg extender. Bioxcell was prepared according to manufacturer s instructions (IMV, France) Semen collection and initial evaluation Two consecutive ejaculates were collected using artificial vagina (at 42 C) from five adult Nili-Ravi buffalo bulls (Bubalus bubalis) of known fertility and similar age (7 8 years) for a period of three weeks (replicate). Ejaculated semen from each bull (6 ejaculates/bull) was immediately transferred to laboratory. Sperm progressive motility was determined microscopically ( 400; Olympus BX20, Tokyo, Japan) with closed circuit television and sperm concentration was determined by Neubauer haemocytometer. At least one ejaculate of each bull at each replicate always passed the criteria (Motility 60%). Qualifying ejaculates from each bull were split into two aliquots and held at 37 C in water bath for 15 min before dilution Semen processing Each semen aliquot from each bull was diluted with one of the two experimental extenders at a concentration of motile spermatozoa ml 1 at 37 C. Diluted semen was cooled to 4 C in 2 hours, equilibrated for 4h at 4 C, filled in 0.5 ml French straws (IMV, France) with suction pump at 4 C in a cold cabinet unit (Minitub, Germany) and placed in liquid nitrogen vapors, 5 cm above the level of liquid nitrogen for 10 min. Straws were then plunged and stored into liquid nitrogen ( 196 C). After 24 h, semen straws were thawed in a water bath at 37 C for 30 seconds [16] Semen quality assays Sperm progressive motility A drop of semen was placed on a pre-warmed microscope slide and subjectively assessed at 37 C for percent progressive motility using phase contrast microscopy (X400; Olympus BX20, Tokyo, Japan) Sperm viability Viability of spermatozoa was assessed with 0.4% Trypan blue stain as described by Brito et al., [17]. A smear of semen (5 l) and equal volume of 0.4% Trypan blue stain was made on a microscope slide and allowed to air dry, before examining under a phase contrast microscope (X1000; Olympus BX20, Tokyo, Japan). One hundred spermatozoa were counted from each sample; unstained white spermatozoa were categorized as viable and blue stained spermatozoa as non-viable Sperm plasma membrane integrity Sperm plasma membrane integrity was determined using hypo-osmotic swelling (HOS) assay [18]. HOS solution was consisted of 0.73g sodium citrate and 1.35 g fructose dissolved in 100 ml distilled water (osmotic pressure 190 mosmol Kg 1 ). To assess the sperm tail plasma membrane integrity, semen (50 l) was mixed with of HOS solution (500 l) and incubated for 30 minutes at 37 C before examining under phase contrast microscope (X400; Olympus BX20, Tokyo, Japan). One hundred spermatozoa were assessed for swelling characterized by coiled tail indicating functional integrity of tail plasma membrane [19] Sperm acrosomal integrity and abnormalities To assess sperm acrosomal integrity and abnormalities, 100 l of semen sample was fixed in 500 l of 1% formal citrate (2.9 g tri-sodium citrate dihydrate, 1 ml of 37% solution of formaldehyde, dissolved in 100 ml of distilled water); one hundred spermatozoa were examined with a phase contrast microscope (X1000; Olympus BX20, Tokyo, Japan) under oil immersion. Normal acrosome was characterized by normal apical ridge. Sperm abnormalities were recorded viz: head abnormalities like micro- and macro-heads, detached heads, double heads, pyriform heads; mid piece abnormalities including prox-

3 S. Akhter et al. / Theriogenology 74 (2010) Table 1 Effect of tris-citric egg and Bioxcell extenders on the post thaw quality (Mean SE; motility, viability, plasma membrane integrity and normal apical ridge) of buffalo bull spermatozoa. Sperm (%) Bull no. Extender imal droplet, distal droplet and abaxial attachment; tail abnormalities like tail coiled below the head, tail bent at mid piece, tail without head and double tail [19] In vivo fertility A total of 400 inseminations were performed with cryopreserved buffalo semen in tris-citric egg and Bioxcell extender from two buffalo bulls, in District Sahiwal, Pakistan. The inseminations were preformed over three months during the peak breeding season. All the experimental inseminations were performed approximately 24 hours after onset of heat. The artificially bred animals were examined for pregnancy through rectal palpation at least 90 days post-insemination under field conditions Statistical analysis Tris-citric egg Bioxcell Motility Overall Viability Overall Plasma membrane integrity Overall Normal apical ridge Overall The data on semen quality parameters were analyzed using t-test and are presented as mean ( SE). A 5% (P 0.05) level was used to determine statistical significance. The data on in vivo fertility rates were analyzed using Chi-square test. 3. Results 3.1. Comparison of post-thaw sperm motility, viability, plasma membrane integrity and acrosomal ridge in tris-citric egg and Bioxcell extenders The data on motility, viability, plasma membrane integrity and acrosomal ridge of buffalo bull spermatozoa are given in Table 1. Percentage of post-thaw sperm motility ( , ), viability ( , ), plasma membrane integrity ( , ) and normal apical ridge ( , ) remained similar (P 0.05) in tris-citric egg and Bioxcell extender, respectively Comparison of post-thaw sperm abnormalities in tris-citric egg and Bioxcell The data on post-thaw abnormalities of buffalo bull spermatozoa are given in Table 2. There was no difference (P 0.05) in sperm abnormalities of head ( , ), mid piece ( , ) and tail ( , ) in tris-citric egg and Bioxcell extender, respectively Comparison of in vivo fertility rates in tris-citric egg and Bioxcell The data on in vivo fertility rates of buffalo semen are given in Table 3. There was no difference (P 0.05) in fertility rates of buffalo semen cryopreserved Table 2 Effect of tris-citric egg and Bioxcell extender on the post thaw abnormalities (Mean SE; head, mid piece and tail) of buffalo bull spermatozoa. Sperm abnormalities (%) Bull no. Extender Tris-citric egg Bioxcell Head Overall Mid piece Overall Tail Overall

4 954 S. Akhter et al. / Theriogenology 74 (2010) Table 3 Effect of tris-citric egg and Bioxcell extender on the fertility rates of frozen buffalo bull spermatozoa. Bull No. Extender No. of inseminations performed in tris-citric egg and Bioxcell extender (44% vs. 47%). 4. Discussion Pregnancies Achieved and Rothe [24] found that soya lecithin based extenders AndroMed, Biociphos Plus and Bioxcell are more capable to maintain higher percentage of live spermatozoa compared to egg extenders. It is pertinent to mention that in soya lecithin based extenders Biociphos Plus, AndroMed and Bioxcell acrosome morphology did not differ [24]. In our study, percentage of normal sperm acrosomes was similar in Bioxcell and tris-citric egg extenders. However, Amirat et al. [10] reported higher sperm numbers with functionally intact acrosomes cryopreserved in soya lecithin based extender; Biociphos Plus as compared to Triladyl an egg based extender and suggested that presence of higher calcium ions of egg might be responsible for the acrosomal damage. Moreover, egg was reported to have some factors which can destabilize the sperm plasma membrane and cause capacitation [25,26]. Muller-Schlosser et al. [23] believed that steroid hormones and its precursors present in egg were associated with poor fertilization potential of the spermatozoa. In this study, post thaw sperm head, mid-piece and tail abnormalities did not differ in all experimental extenders. It was observed that preservation of buffalo semen did not increase the sperm abnormalities [19]. It was recently reported that in the domestic bull Bioxcell has the ability to maintain glutathione content in cryopreserved sperm because it contains higher concentrations of glutathione than tris-egg extender (450 umoles vs 40 umoles) [14]. As a positive relationship has been reported between glutathione levels and semen quality [14,27], it is suggested that improvement of post-thaw quality of bovine semen [8,14] cryopreserved in Bioxcell may be attributed to the higher concentrations of glutathione which protect the sperm against oxidative stress and reactive oxygen species. However, for buffalo semen Bioxcell could not improve semen quality as compared to tris-citric egg extender. It is suggested that buffalo bull spermatozoa is more prone to oxidative stress due to high contents of polyunsaturated phospholipids [2]; therefore, the level of glutathione in Bioxcell is insufficient to protect the sperm from oxidative stress during freeze-thawing process. In the present study similar fertility rates were recorded of buffalo semen cryopreserved in tris-citric egg extender and Bioxcell. In similar study on ram semen, equivalent fertility rates with frozen semen were recorded using Bioxcell and egg extenders [12]. Although Bioxcell produced fertility results equivalent to routinely used tris-citric egg ex- Chisquare 1 Tris-citric egg (45%) 0.18 NS Bioxcell (45%) 0.18 NS 2 Tris-citric egg (43%) Bioxcell (46%) 0.36 NS Overall Tris-citric egg (44%) Bioxcell (47%) NS, Non Significant. The present study was designed to compare the post thaw semen quality (motility, viability, plasma membrane integrity, normal apical ridge and abnormalities) of cryopreserved buffalo semen using commercially available extender Bioxcell and tris-citric egg extender. In the present study, post thaw motility of buffalo spermatozoa was similar in tris-citric egg and Bioxcell extender. In similar studies on ovine semen [11,12], similar post-thaw sperm motilities were recorded in Bioxcell and milk based extender. However, Stradaioli et al. [14] observed higher post thaw motility of bovine spermatozoa in Bioxcell compared to trisegg extender. It was suggested that higher sperm motility in Bioxcell might be due to higher content of glutathione in Bioxcell as compared to tris-egg extender. Similarly, in soya lecithin based prepared extenders; Biociphos Plus [8,10,20,21] and AndroMed [3], higher motility of bovine sperm was observed as compared to egg based extenders. Post thaw viability and plasma membrane integrity of buffalo spermatozoa was similar in Bioxcell and tris-citric egg extender in our study. Higher viability of bovine spermatozoa has previously been observed in soya lecithin based extender, Biociphos Plus [10] and Bioxcell [8] compared to Triladyl an egg based extender. It is believed that egg can change the sperm chromatin structure which result in poor post-thaw sperm viability [11,12,22]. Muller- Schlosser [23] suggested that inconsistent egg composition is detrimental to sperm viability. Nehring

5 S. Akhter et al. / Theriogenology 74 (2010) tender but it has advantage in terms of lowest sanitary risks, chemically defined and ready to use. It is concluded that commercially available Bioxcell may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg extender. Acknowledgements The authors thank Pakistan Science Foundation for the financial support under the Natural Sciences Linkage Programme and to anonymous reviewers for their valuable comments and suggestions. References [1] Andrabi SMH. Factors affecting the quality of cryopreserved buffalo (bubalus bubalis) bull spermatozoa. Reprod Domest Anim 2009;44: [2] Sansone G, Nastri MJF, Fabbrocini A. Storage of buffalo (Bubalus bubalis) semen. Anim Reprod Sci 2001;62: [3] Aires VA, Hinsch KD, Mueller-Schloesser F, Bogner K, Mueller- Schloesser S, Hinsch E. In vitro and in vivo comparison of egg -based and soybean lecithin-based extenders for cryopreservation of bovine semen. 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