Evaluation of soya lecithin based extender for liquid storage of Nili-Ravi buffalo (Bubalus bubalis)

Abstract

The present study was designed to compare the soya lecithin based extender Bioxcell® (BIOX) with milk (MILK), tris-egg yolk (TEY) and egg yolk-citrate (EYC) extender for the liquid storage of buffalo bull spermatozoa at 5°C. Semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina over a period of 3 weeks. Qualifying semen ejaculates were pooled, split into four aliquots, diluted (37oC; 10 x 106 motile spermatozoa per ml), cooled from 37oC to 5oC in 2 hours and stored for five days. Sperm motility, viability, plasma membrane integrity (PMI), normal acrosomal ridge (NAR) and abnormalities (head, mid piece and tail abnormalities) were studied at 1st, 3rd and 5th day of storage. Higher values of progressive sperm motility (%), sperm viability (%), sperm plasma membrane integrity (PMI, %) and NAR (%) were observed in BIOX, MILK, and TEY extenders at 1st, 3rd and 5th day of storage than EYC extender. Progressive sperm motility, sperm viability and sperm plasma membrane integrity was not affected up to three days storage period in BIOX extender and at 5th day of storage the values for these parameters remained significantly higher in BIOX than MILK, TEY and EYC extenders. Sperm abnormalities (head, mid piece and tail) did not differ (P > 0.05) due to extenders at 1st, 3rd and 5th day of storage. In conclusion, Bioxcell® is more capable than milk, tris-egg yolk and egg yolk-citrate extender for maintaining the quality of buffalo bull spermatozoa stored at 5 °C.

Keywords:

Bioxcell®; buffalo bull; liquid storage

1. Introduction

Artificial insemination (AI) allows rapid dissemination of genetic material from a small number of superior sires to a large number of females (Vishwanath and Shannon, 1997) and semen from farm animals used for this purpose can been stored either for short-term at 4C in the liquid form or for long term in cryopreserved state in liquid nitrogen (-196C) (Shannon 1978; Johnson et al. 1981; Weitze, 1991; Maxwell and Watson 1996). Cryopreservation of bovine semen is practiced worldwide for many reasons but the use of liquid semen for AI has its own advantages. The main benefit of liquid semen is that a comparatively higher conception rate is achieved (Anzar et al., 2003; Sharma and Sahni, 1988; Shannon and Vishwana, 1995) with relatively lower number of spermatozoa (Vishwanath et al., 1996).

Commonly, buffalo semen is preserved in milk ((Kumar et al., 1993; El-Azab, 1998; Pramanik et al., 1998; Akhter et al., 2008), tris-egg yolk (Rasul et al., 2000 and 2001; Andrabi et al., 2008) and egg yolk-citrate (Sansone et al., 2000) extenders. These extenders contain additives of animal origin (egg yolk and/or milk) which may pose a potential risk of microbial contamination (Bousseau et al., 1998; Thibier and Guerin et al., 2000). Presence of bacteria in the inseminate has been reported to, deteriorate its quality (Aleem et al., 1990; Dela Pena et al., 1995; Akhter et al., 2008), cause reproductive problems in female and result in low fertility rate (Andrabi et al., 2001; Akhter et al., 2007). Therefore, a well defined and pathogen-free substitute for egg yolk (preferably of non-animal origin) would be more suitable for extenders used for semen (Stradaioli et al., 2007).

A number of commercially available extenders containing a substitute for egg yolk have been used for the preservation of bovine, ovine and caprine semen by a number of workers. The examples are of AndroMed® (Aires et al., 2003; Nagy et al., 2003; Herold et al., 2004; Herold et al., 2006; Muino et al., 2007), Biociphos plus ® (Haard, 1997; Gil et al., 2000; Leeuw et al., 2000; Thun et al., 2002; Nagy et al., 2003; Amirat et al., 2005; Muino et al., 2007), and Bioxcell® (Gil et al., 2003a,b; Hansen et al., 2005; Stradaioli et al., 2007; Celeghini et al., 2008). Preservation of bull (Hansen et al., 2005; Stradaioli et al., 2007; Celeghini et al., 2008) and ram (Gil et al., 2003a,b) spermatozoa in soya lecithin based extender Bioxcell® maintained the sperm quality and produced acceptable fertility rates (Gil et al., 2003b). To our knowledge, not a single study is available on the liquid preservation of Nili-Ravi buffalo semen in prepared extenders containing soya lecithin as an egg yolk substitute. Hence, present study was designed to evaluate and compare the soya lecithin based extender, Bioxcell®, with milk, Tris-egg yolk and egg yolk-citrate extenders for liquid storage of buffalo bull spermatozoa at 5°C.

2. Materials and Methods

2.1. Preparation of extenders

Four experimental extenders, i.e., tris-egg yolk (TEY) (1.56 g citric acid (Fisher Scientific, UK) and 3.0 g tris-(hydroxymethyl)-aminomethane (Research Organics, USA); 80 ml distilled water; fructose (Scharlau, Spain) 0.2% wt/vol; egg yolk 20%), egg yolk-citrate (EYC) (2.9 g sodium citrate (Applicham, Germany); 80 ml distilled water; fructose 0.2% wt/vol; egg yolk 20% v/v), milk extender (MILK) (10 % skim milk powder, Haleeb®) and Bioxcell (prepared according to manufacturer instructions, IMV, France) were used in this study.
Antibiotics including gentamycin sulphate (500 µg/ml; Reckitt Benckiser, Pakistan), tylosin tartrate (100 µg/ml; VMD, Belgium), lincomycin hydrochloride (300 µg/ml; Pharmacia & Upjohn, Belgium) and spectinomycin hydrochloride (600 µg/ml; Pharmacia & Upjohn, Belgium) were added to TEY, EYC and MILK extenders.

2.2. Semen collection and initial evaluation

Using artificial vagina (42°C) semen was collected (two consecutive ejaculates/bull/week) from 5 adult Nili-Ravi buffalo bulls (Bubalus bubalis) maintained at the Semen Production Unit Qadirabad, Sahiwal, Pakistan for a period of 3 weeks. The semen was transferred to laboratory within minutes of collection. Visual motility was assessed microscopically (at 200x) with closed circuit television (Graham et al., 1970) and sperm concentration was determined by Neubauer haemocytometer. The neat semen samples qualifying a minimum standard of 1 ml volume, 60% motility and with a concentration of 0.5 billion sperms/ml of the ejaculate were selected for further processing. In order to have sufficient semen for a replicate and to eliminate the bull effect, the qualifying ejaculates were pooled and held for 15 min at 37 °C in a water bath before dilution. Pooled semen was split into four equal aliquots for dilution in four different experimental extenders.

2.3. Semen processing

Semen aliquots were diluted at 37°C with one of the four experimental extenders at the rate 10 × 106 motile spermatozoa ml-1 approximately. Diluted semen was cooled to 5°C in 2 hours at the rate of 0.275 °C min-1 and stored for five days at 5°C

2.4. Sperm functional assays

Sperm quality assays including sperm progressive motility, sperm viability (Live/dead percentage), sperm plasma membrane integrity, sperm acrosomal integrity and sperm abnormalities were performed at 1st, 3rd and 5th day of storage. Briefly, for sperm progressive motility, a drop of semen sample was placed on pre-warmed glass slide, cover-slipped and progressive motility was assessed at 37°C with phase contrast microscope (X 400). Sperm viability (Live/dead percentage) was assessed using 0.4% Trypan blue stain according to Brito et al. (2003). For this purpose, 5µl semen sample and an equal amount of Trypan Blue solution were mixed together. Using this mixture a smear was made on a glass slide, air dried and examined under phase contrast microscope (X 1000; oil immersion). Spermatozoa stained blue were considered as dead while unstained as live. A total of one hundred spermatozoa were counted. Sperm plasma membrane integrity (PMI) was assessed by hypo-osmotic swelling (HOS) assay (Jeyendran et al., 1984). Solution for HOS assay consisted of 0.73g sodium citrate and 1.35g fructose dissolved in 100ml distilled water (osmotic pressure ~190 mOsmol Kg-1). For the assessment of sperm tail plasma membrane integrity 50ml of semen was mixed with 500ml of HOS solution and incubated for 30-40 min at 37°C. A drop of semen sample was placed on a glass slide and cover-slipped to examine under phase contrast microscope (X 400). One hundred spermatozoa were examined for their swelling characterized by coiled tail indicating intact sperm plasma membrane (Ahmed et al., 2003). For sperm acrosomal integrity and abnormalities semen samples (100µl) was fixed in 500µl of 1% formal citrate (2.9 g tri-sodium citrate dehydrate, 1 ml of 37% solution of formaldehyde, dissolved in 100 ml of distilled water) and one hundred spermatozoa were examined with a phase contrast microscope (X 1000) under oil immersion. Normal acrosome was characterized by normal apical ridge (NAR). Sperm abnormalities recorded were micro- and macro-heads, detached heads, double heads, pyriform heads, mid piece abnormalities including proximal droplet, distal droplet and abaxial attachment and tail abnormalities like tail coiled below the head, tail bent at mid piece, tail without head and double tail.

2.5. Statistical analysis

The data are presented as mean (± SD) values of different parameters used to assess the quality of the semen samples. Data were analyzed by ANOVA using a 3 x 4 factorial design. When appropriate, Post-hoc comparisons for treatment and time were performed using LSD. A level of 5% (P < 0.05) was used to determine statistical significance (MSTAT-C Ver.1.42).

3. Results

Progressive sperm motility (Figure 1), sperm viability (Figure 2), sperm plasma membrane integrity (Figure 3) and NAR (Figure 4) were significantly (P < 0.05) higher for BIOX, MILK, and TEY extenders at 1st, 3rd and 5th day of storage compared to EYC extender. Moreover, progressive sperm motility, sperm viability and sperm plasma membrane integrity were not affected upto third day of storage in BIOX extender and at 5th day of storage the values for these parameters remained significantly (P < 0.05) higher in BIOX compared to other extenders. Sperm abnormalities (head, mid piece and tail) did not differ among the different extenders at 1st, 3rd and 5th day of storage.

4. Discussion

This study was designed to evaluate a soya lecithin based extender, Bioxcell®, for liquid storage of buffalo bull spermatozoa at 5°C and its comparison with milk, Tris citric acid and sodium citrate based extenders.

Sperm motility is an indicator of sperm quality and is commonly used in the laboratory. Chemical and physical properties of the extender affect the sperm motility (Rasul et al. 2000). In our study, sperm motility was found higher in Bioxcell® for five days of storage at 5 °C. A higher, although non-significantly different, sperm motility has been reported for Bioxcell® as compared to milk extender for ram semen (Gil et al., 2003a,b). Using soya lecithin based extender Biociphos Plus® frozen-thawed semen is also reported to have higher percentage of sperm motility as compared to egg yolk based prepared extender Triladyl® (Gil et al., 2000; Moussa et al., 2002; Amirat et al., 2004; Amirat et al., 2005). Studies by Aries et al. (2003) have also reported a significant increase in post-thaw sperm motility in soya lecithin based extender AndroMed® as compared to egg yolk extender. Recently, in a study on bovine semen (Stradaioli et al., 2007) higher sperm motility of spermatozoa cryopreserved in Bioxcell® as compared to tris-egg yolk extender has been reported. Low density lipoproteins protect the sperm during freezing while high density lipoprotein contents of egg yolk deteriorate the quality of the sperm (Amirat et al., 2005) and deleterious effect of egg yolk results in reduction of respiration and sperm motility (Kmpshmidt et al., 1953; Pace and Graham, 1974; Watson and Martin, 1975). Gil et al., (2003a,b) believed that non-animal substitute of milk or egg yolk in an extender results in improved bacteriological qualities which protect the sperm from toxins produced by bacteria in the semen.

The number of viable sperms per insemination dose after cryopreservation significantly affects fertility rates in the field (Andrabi et al., 2006). In our study, sperm viability was found higher in Bioxcell for five days of storage at 5 °C. Amirat et al., (2005) reported higher viable sperms in soya lecithin based extender, Biociphos Plus® as compared to egg yolk based extender Triladyl®. Gil et al. (2000) also reported a higher percentage of bull sperms with intact plasma membrane as compared to egg yolk based extender. Milk and egg yolk in extender can alter the sperm chromatin structure which result in poor post-thaw viability (Karabinus et al., 1991; Gil et al., 2003a,b). Moreover, the quality of the egg yolk is not consistent and has deleterious effect for sperm viability (Muller-Schlosser, 2001). Similar results were found in studies on bovine spermatozoa preserved with soya lecithin based extenders AndroMed®, Biociphos Plus® and Bioxcell® in which higher percentage of live spermatozoa was recorded as compared to egg yolk based extenders (Nehring and Rothe, 2003).

The evaluation of plasma membrane integrity is of significance as it controls the metabolic exchanges with the surrounding medium (Silva and Gadella, 2006) and a biochemically active plasmalemma is required for the process of capacitation, acrosome reaction and the oocyte penetration (Jeyendran et al., 1984). Hypo-osmotic swelling test has been widely used to assess the plasma membrane functional intactness in buffalo bull spermatozoa (Rasul et al., 2001; Akhter et al., 2008). In our study, sperm with intact plasma membrane was found higher in Bioxcell® for five days of storage at 5 °C. Gil et al. (2003a,b) reported a non significant difference in sperm plasma membrane integrity of ovine semen in Bioxcell® as compared to milk extender. Some factors in egg yolk are reported to destabilize the sperm plasma membrane during storage (Watson and Martin, 1976; Smith et al., 1979). It is noteworthy that the data on the plasma membrane integrity support the sperm progressive motility.

The presence of normal acrosome on a spermatozoon is essential for the acrosomal reaction required to facilitate fertilization (Thomas et al., 1997) and its assessment can be an effective tool to predict the fertilizing ability of buffalo bull spermatozoa. The change in acrosomal cap is mainly due to sperm aging or cryo-injury, which can be effectively determined by fixing the specimen using phase contrast microscope (Anzar and Graham, 1993). A high correlation between the percentage of intact acrosome and fertility of frozen bovine spermatozoa was reported after 2 and 4 h of post-thaw incubation (Saacke and White, 1972). In our study, sperm with intact acrosomes was found higher in Bioxcell® for five days of storage at 5 °C. Amirat et al. (2005) reported 30-80 % sperm acrosomal damage after one hour of dilution in egg yolk based extender Triladyl®, and suggested that higher calcium ions present in the egg yolk cause acrosomal destruction especially below 30 °C, because of higher rate of calcium ions infusion. In egg yolk some factors are reported to destabilize the sperm plasma membrane and cause capacitation (Watson and Martin, 1976; Smith et al., 1979). The reduction in fertilizing ability of the sperm is also associated with the steroid hormone and it precursor molecules present in egg yolk (Muller-Schlosser et al., 2001). Gil et al. (2003a,b) reported high percentage of uncapacitated sperm and lower sperm with reacted acrosome in Bioxcell® as compared to milk extender of ram semen. Gil et al. (2000) also reported a higher percentage of sperm with intact plasma membrane as compared to egg yolk based extender in bovine semen.

Evaluation of sperm abnormalities is one of the commonest methods to assess functional status of the semen (Rocha et al., 2006; Sajjad et al., 2007; Akhter et al., 2008). A significant correlation has also been established between the fertility of frozen-thawed bull semen and some sperm abnormalities like sperm head forms and the presence of proximal cytoplasmic droplets (Soderquist et al., 1991). In our study, post-thaw sperm head, mid-piece and tail abnormalities were similar in all the four experimental extenders. It is relevant that semen processing does not necessarily increase abnormal heads, detached heads, coiled tails and proximal cytoplasmic droplets/pseudodroplets in bovine (Revell, 2003) and buffalo semen (Akhter et al., 2008). It is worth to highlight that in egg yolk free soya lecithin based extenders, morphology of acrosome did not differ after preservation and storage in either Biociphos Plus® or AndroMed® or Bioxcell® (Nehring and Rothe, 2003).

5. Conclusion

In conclusion, Bioxcell® is more capable than milk, tris-egg yolk and egg yolk-citrate extender for maintaining the quality of buffalo bull spermatozoa stored at 5 °C. It can be recommended for routine liquid preservation of buffalo semen for artificial insemination programmes to avoid the potential risks of contamination from constitutes of animal origin.

References

Ahmad, Z., Anzar, M., Shahab, M., Ahmad, N., Andrabi, S.M.H., 2003. Sephadex and sephadex ion exchange filtration improves the quality and freezability of low-grade buffalo semen ejaculates. Theriogenology 59, 1189-1202.

Aires, V.A., Hinsch, K.D., Mueller-Schloesser, F., Bogner, K., Mueller-Schloesser, S., Hinsch, E., 2003. In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen. Theriogenology 60, 269-279.

Akhter S., Sajjad, M., Andrabi, S.M.H., Ullah, N., Qayyum, M., 2007. Effect of antibiotics in extender on fertility of liquid buffalo bull semen. Pakistan Vet. J. 27 (1), 13-16.
Akhter, S., Ansari, M.S., Andrabi, S.M.H., Ullah, N., Qayyum, M., 2008. Effect of Antibiotics in Extender on Bacterial and Spermatozoal Quality of Cooled Buffalo (Bubalus bubalis) Bull Semen. Reprod. Dom. Anim. 43, 272-278.

Aleem, M., Chaudhry, R.A., Khan, N.U., Rizvi, A.R., Ahmed, R., 1990. Occurrence of pathogenic bacteria in buffalo semen. Buffalo J. 6, 93-98.

Amirat, L., Anton, M., Tainturier, D., Chatagnon, G.R., Battut, I., Courtens, J.L., 2005. Modifications of bull spermatozoa induced by three extenders: Biociphos, low density lipoprotein and Triladyl, before, during and after freezing and thawing. Reprod. 129, 535-543.

Amirat, L., Tainturier, D., Jeannaau, L., Thorin, C., Gerard, O., Counters, J.L., Anton, M., 2004. Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl, a commercial egg yolk extender. Theriogenology 61, 895-907.

Andrabi, S.M.H., Ahmad, N., Abbas, A., Anzar, M., 2001. Effect of two different antibiotic combinations on fertility of frozen buffalo and Sahiwal bull semen. Pakistan Vet. J. 21, 166-169.

Andrabi, S.M.H., Ansari, M.S., Ullah, N., Anwar, M., Mehmood, A., Akhter, S., 2008. Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa. Anim. Reprod. Sci. 104, 427-433.

Andrabi, S.M.H., Siddique, M., Ullah, N., Khan, L.A., 2006. Effect of two different antibiotic combinations on fertility of frozen buffalo and sahiwal semen. Pakistan Vet. J. 26, 17-19.

Anzar, M., Farooq, U., Mirza, M.A., Shahab, M.., Ahmad, N., 2003. Factors affecting the efficiency of artificial insemination in cattle and buffalo in Punjab, Pak. Vet. J. 23, 106-113.

Anzar, M., Graham, E.F., 1993. Filtration of bovine semen. I. Development of a Sephadex-ion exchange filter. Anim. Reprod. Sci. 31, 187-195.

Bousseau, S., Brillard, J.P., Marquant, L., Guienne, B., Guerin, B., Camus, A., Lechat, M., 1998. Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. Theriogenology 50, 699-706.

Brito, L. F.C., Albert D. B., Sylvie B. G., Paul L. P., John P. K., 2003. Comparison of methods to evaluate the plasmalemma of bovine sperm and their relationship with in vitro fertilization rate. Theriogenology 60, 1539-1551.

Celeghini, E.C.C., Arruda, R.P.D., Andrade, A.F.C.D., Nascimento, J., Raphael, C.F., Roderigues, P.H.M., 2008. Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin. Anim. Reprod. Sci. 104, 119-131.

Dela Pena, E.C., Limcumpao, J.A., Bain, C.B., Vizmanos, E.C., Momongan, V.G., Palad, O.A., 1995. Study on sperm motility and bacterial counts in buffalo semen stored at various temperatures. Philippines J. Vet. Anim. Sci. 21, 53-66.

El-Azab., Khadr, N.A., Zahran, K., 1998. Effect of non-protein nitrogen in the ration on ram semen quality A.I. Small Rumi. Res. 27, 73-77.

Gil, J., Januskausks, A., Haard, M.C., Haard, M.G.M., Johanisson, A., Soderquist, L., Rodriguez-Martinez, H., 2000. Functional sperm parameters and fertility of bull semen extended in Biociphos Plus. Reprod. Dom. Anim. 35, 69-77.

Gill, J., Lundeheim, N., Soderquist, L., Rodriguez-Martinez, H., 2003a. Influence of extender, temperature, and addition of glycerol on post-thaw sperm parameters in ram semen. Theriogenology 59, 1241-1255.

Gil, J., Rodriguez-Irazoqui, M., Lundeheim, N., Soderquist, L., Roderiguez-Martinez, H., 2003b. Fertility of ram semen in Bioexcell and used for cervical artificial insemination. Theriogenoloy 59, 1157-1170.

Graham, E.F., Schmehl, M.K.L., Maki-Laurila, M., 1970. Some physical and chemical methods of evaluating semen. In: Proceedings of the 3rd National Association of Animal Breeders Technical Conference on Artifical Insemination and Reproduction. Milwaukee, WI. 44-48.

Gupta, H.P., Tripathi, S.S., 1984. Preservation of semen from red dane bull with different room temperature dilutors. Indian J. Anim. 54, 494-497.

Haard, M., 1997. Semen processing general aspects new diluents cooling rates Proceedings of 8th European AI Vets Meeting. Switzerland. 18-22.

Hansen, G.B.B., Morris, I.D., Ersboll, A.K., Greve, T., Christensen, P., 2005. DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay.Theriogenology 63, 1789-1802.

Herold, F.C., Aurich, J.E., Gerber, D., 2004. Epididymal sperm from the African buffalo (Syncerus caffer) can be frozen successfully with andromed and with Triladyl but the addition of bovine seminal plasma is detrimental. Theriogenology 61, 715-724.

Herold, F.C., D.H.K., Colenbrander, B., Gerber, D., 2006. Comparison of equilibration times when freezing epididymal sperm from African buffalo (Syncerus caffer) using Triladyl TM or AndroMed. Theriogenology 66, 1123-1130.

Jeyendran, R.S., Van Der Ven, H.H., Perez-Pelaez, M., Crabo, B.G., Zaneveld, L.J.D., 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Ferti. 70, 219-228.

Johnson, A., Aalbers, J.G., Willems, C.M.T., Sybesma, W., 1981. Use of Boar Spermatozoa for Artificial Insemination. I. Fertilizing Capacity of Fresh and Frozen Spermatozoa in Sows on 36 Farms. J. Anim. Sci. 52, 1130-1136.

Kampshmidt, R.F., Mayer, D.T., Herman, H.A., 1953. Lipid and lipoprotein constituents of egg yolk in the resistance and storage of bull spermatozoa. J. Dairy. Sci. 36, 733-742.

Karabinus, D.S., Evenson, D.P., Kaproth, M.T., 1991. Effects of egg yolk-citrate and milk extenders on chromatin structure and viability of cryopreserved bull sperm. J. Dairy Sci. 74, 3836 -3848.

Kumar, S., Sahni, K.L., Bistha, G.S., 1993, Cytomorphological characteristics of motile and static semen of buffalo bulls. Buffalo J. 2, 117-127.

Leeuw, V.W., Haring, A.M., Kaal-Lansbergen, R.M., LMTE, D.D., 2000. Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soy bean extract. Theriogenology 54, 57-67.

Maxwell, W.M.C., Watson, P.F., 1996. Recent progress in the preservation of ram semen. Anim. Reprod. Sci. 42, 55-65.

Meister, A., Anderson, M.E., 1983. Glutathione. Ann. Rev. Biochem. 52, 11-60.
Moussa, M., Martiner, V., Trimeeche, A., Tainturier, D., Anton, M., 2002. Low density lipoproteins extracted from hen egg yolk by an easy method: cryopreservative effect on frozen-thawed bull semen, Theriogenology 57, 1695-1706.

Muino, R., Fernandez, M., Pen, A.I., 2007. Post-thaw Survival and Longevity of Bull Spermatozoa Frozen with an Egg Yolk-based or Two Egg Yolk-free Extenders after an Equilibration Period of 18 h. Reprod. Dom. Anim. 42, 305-311.

Muller-Schlosser, F., Aires, V., Hinsch, E., Hinsch, K.D., 2001. Evaluation of the quality of a new generation of egg yolk free semen diluters for cryopreservation of bovine semen. 34th Conference on physiology and pathology of reproduction, Giessen..

Nagy, J., Jansen, J., Topper, E.K., Gadella, B.M., 2003. A triple-stain flow cytometric method to assess plasma and acrosome membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg yolk particles. Biol. Reprod. 68, 1828-1835.

Nehring, H., Rothe, L., 2003. Insemination of cryopreserved bull semen portions with reduced sperm numbers after dilution with two egg yolk-free extenders. Proc. 15th Europ. AI Vets Meet. Budapest Hungary, 14-23.

Pace, M.M., Graham, E.F., 1974. Components in egg yolk which protect bovine spermatozoa during freezing. J. Anim. Sci. 39, 1144-1149.

Pramanik, P.S., Raina, V.S. ,1998. Refrigerator (4-7ºC) preservation of buffalo semen in various extenders. Indian J. Dairy Sci. 51, 375-379.

Rasul, Z., Ahmad, N., Anzar, M., 2001. Changes in motion characteristics, plasma membrane integrity, and acrosome morphology during cryopreservation of buffalo spermatozoa. J. Andrology 22, 278-283.

Rasul, Z., Anzar, M., Jalali, S., Ahmad, N., 2000. Effect of buffering system on post-thaw motion characteristics, plasma membrane integrity and acrosome morphology of buffalo spermatozoa. Anim. Reprod. Sci. 59, 31-41.

Revell, S.G., S, B.M.V., S, M.R.C.V., Cranfield, N., Cooley, W., 2003. Sperm tail-stump defect in a Holstein bull. British Vet. Associ. 153(9), 268-268.

Roche, J.R., Lee, J.M., Berry, D.P., 2006. Climatic Factors and Secondary Sex Ratio in Dairy Cows. J. Dairy Sci. 89, 3221-3227.

Saacke, R.G., White, J.M., 1972. Semen quality tests and their relationship to fertility. Proceedings of the 4th Technical Conference on Artificial Insemination and Reproduction, N A A B. 22-27.

Sajjad, M., Ali, S., Ullah, N., Anwar, M., Akhter, S., Andrabi, S.M.H., 2007. Blood serum testosterone level, its relationship with scrotal circumference and semen characteristics in Nili-Ravi buffalo bulls. Pakistan Vet. J. 27 (2), 63-66.

Sansone, G., Nastri, M.J.F., Fabbrocini, A., 2000. Storage of buffalo (Bubalus bubalis) semen. Anim. Reprod. Sci. 62, 55-76.

Shannon, P., 1973. Factors affecting storage of semen. Proc. NZ. Soc. Anim. Prod. 33, 40-48.

Shannon, P., 1978. Advances in semen dilution. Proc. Soc. Anim. Prod. N.Z. 28, 23-31.

Shannon, P., Vishwanath, R., 1995. The effect of optimal and suboptimal concentrations of sperm on the fertility of fresh and frozen bovine semen and a theoretical model to explain the fertility differences. Anim. Reprod. Sci. 39, 1-10.

Sharma N.C., Sahni, K.L., 1988. Fertility of frozen and liquid semen in buffaloes. World Buffalo Congress, 100.

Silva, P.F.N., Gadella, B.M., 2006. Detection of damage in mammalian sperm cells. Theriogenology 65, 958-978.

Smith, R.L., Berndston, W.E., Unal, M.B., Pickett, B.W., 1979. Influence of percent egg yolk during cooling and freezing on survival of bovine spermatozoa. J. Dairy Sci. 62, 1297-1303.

Soderquist, L., Janson, L, Larsson, K., Einarsson, S., 1991. Sperm morphology and fertility in A.I. bulls. J. Vet. Med. 38, 534-543.

Stradaioli, G., Noro, T., Sylla, L., Monaci, M., 2007. Decrease in glutathione (GSH) content in bovine sperm after cryopreservation: comparison between two extenders. Anim. Reprod. Sci. 67, 1249-2155.

Thibier, M., Guerin, B., 2000. Hygienic aspects of storage and use of semen for artificial insemination. Anim. Reprod. Sci. 62, 233-251.

Thomas, C.A., Garner, D.L., de-Jarnette, J.M., Marshall, C.E., 1997. Fluorometric assessment of acrosomal integrity and viability in cryopreserved bovine spermatozoa. Biol. Reprod. 45, 880-887.

Thun, R., Hurtado, M., Janett, F., 2002. Comparison of Biociphos Plus and Tris-egg yolk extender for cryopreservation of bull semen. Theriogenology 57, 1087-1094.

Vishwanath, R., Pitt, C.P., Shannon, P., 1996. Sperm numbers, semen age and fertility in fresh and frozen bovine semen. Proc. N.Z. Soc. Anim. Prod. 56, 31-34.

Vishwanath, R., Shannon, P., 1997. Do sperm cells age? A review of the physiological changes in sperm during storage at ambient temperature. Reprod. Fertil. Dev. 9, 321-332.

Watson, P.F., Martin, C.A., 1975. The influence of some fractions of egg yolk on the survival of ram spermatozoa at 5oC. Australian J. Bio. Sci. 28, 145-152.

Watson, P.F., Martin, I.C., 1976. Artificial insemination of sheep: the effect of semen diluents containing egg yolk on the fertility of ram semen. Theriogenology 6, 927-935.

Weitze, K.F., 1991. Long term storage of extended boar semen in: Johnson LA, 2nd Int conf on Boar semen preservation, USA. Reprod. Dom. Anim. 231-253.