Preservation of mithun (Bos frontalis) semen at refrigeration temperature

Animal Reproduction Science 101 (2007) 257–264

Preservation of mithun (Bos frontalis) semen at refrigeration temperature M. Karunakaran a,b , A. Dhali a,∗ , A. Mech a , K. Khate a , C. Rajkhowa a , D.P. Mishra a,1 a

National Research Centre on Mithun, Medziphema, Nagaland 797 106, India b ICAR-RC-NEH Region, Medziphema, Nagaland 797 106, India

Received 23 May 2006; received in revised form 26 August 2006; accepted 7 September 2006 Available online 9 September 2006

Abstract The objective of the present study was to investigate the possibility of preserving mithun (Bos frontalis) spermatozoa at refrigeration temperature using tris–egg yolk diluent. Semen samples were collected from four adult mithun bulls through rectal massage method. Good quality semen samples (n = 30) were preserved at 4 ◦ C using tris–egg yolk diluent for 72 h. Progressive motility, live spermatozoa count and morphological abnormalities were evaluated every 12 h until 72 h of preservation. The colour, consistency and mass activity of fresh semen samples were found to be creamy white, medium and 3+ to 4+ (5+ scale), respectively. The average (mean ± S.E.) volume (ml), pH and spermatozoa concentration (106 ml−1 ) of fresh semen samples were found to be 0.6 ± 0.01, 6.8 ± 0.03 and 425 ± 48, respectively. Progressive motility and live spermatozoa count were found to be less than 30% (P < 0.01) after 48 h of storage. Head (P < 0.05), midpiece (P < 0.05), tail (P < 0.01) and total (P < 0.01) abnormalities were found to be increased significantly over the time of storage. It was observed that progressive motility and live spermatozoa count remained above 30% and 40%, respectively, until 36 h of storage. Simultaneously the percentage of morphologically abnormal spermatozoa was found to be significantly low until 36 h of storage. The results indicate that it is possible to preserve mithun spermatozoa at refrigeration temperature in tris–egg yolk diluent, which can be further used for artificial insemination within 36 h of storage. © 2006 Elsevier B.V. All rights reserved. Keywords: Spermatozoa; Preservation; Motility; Abnormalities; Mithun

∗ Corresponding author. Present adress: Dairy Science Department, Virginia Polytechnic Institute and State University, 2580 Litton Reaves Hall, Blacksburg, VA 24061, USA. Tel.: +1 540 231 5849. E-mail address: [email protected] (A. Dhali). 1 Department of Pharmacology, 360D, Med Surge II, UC, Irvine, CA 92697, USA.

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.09.011

258

M. Karunakaran et al. / Animal Reproduction Science 101 (2007) 257–264

1. Introduction Mithun (Bos frontalis), the domesticated form of wild gaur, is found mainly in the hilly pockets of South-East Asia at an elevation of 1000–3000 m above sea level. This unique bovine species is primarily used as a beef animal. Besides, it also plays an important role in the socio-economic life of its rearers (Simoons, 1984; Mondal and Pal, 1999). At present, farmers rear this animal under free-range system without much emphasis on scientific breeding and management practices. The recent initiatives to popularize this species as an economic beef animal demand its rearing under semi-intensive system and adoption of controlled breeding programme. In this context, it is necessary to standardize an effective semen preservation protocol for this species to adopt artificial insemination (AI) for breed improvement programme. The wide spread uses of AI in bovine species have become possible due to the availability of suitable diluents and due to the fact that similar pregnancy rates can be achieved with small doses of preserved semen (Verberckmoes et al., 2004). The ability to obtain high pregnancy rates with low insemination doses increases the number of insemination doses produced by genetically superior sires and accelerates the genetic progress in livestock. Cold storage of semen is used to reduce metabolism and to maintain sperm viability over an extended period of time. Researches into extender development have focused on the subject of membrane stabilizing compounds. Egg yolk and milk have been used for long time sperm preservation and been the subject of many investigations (Watson, 1981; Dee Leeuw et al., 1993). It is reported that fresh bovine semen can be preserved successfully at 5 ◦ C for 6 days using tris–egg yolk based extender without glycerol (Verberckmoes et al., 2004). A previous report indicates that cryopreserved gaur semen with 50% motility and 63% morphologically normal spermatozoa is capable of fertilizing Bos taurus oocytes (Hopkins et al., 1988). It is also reported that cryopreserved gaur spermatozoa are capable of establishing pregnancy in cattle and gaur (Hopkins et al., 1988; Godfrey et al., 1991; Sukwongs et al., 1998). Besides, the previous investigations indicate the utilization of gaur semen for in vitro embryo production (Johnston et al., 1994; Hammer et al., 2001). The only report on the characteristics of mithun semen indicates that the different parameters of the semen collected through rectal palpation are within the normal range and can be compared with cattle semen (Bhattacharya et al., 2005). At present, no report is available on the preservation of mithun semen. Moreover, there is no available literature on AI and in vitro embryo production using preserved semen in this species. Therefore, the purpose of the present study was to determine if mithun semen could be preserved at refrigeration temperature using tris–egg yolk based extender. 2. Materials and methods 2.1. Experimental animals and semen collection During the experiment, four healthy adult mithun bulls (4–6 years of age) were used for semen collection. The animals were maintained at the Institute mithun farm, Jharnapani, Nagaland, India. Each experimental animal was daily offered ad libitum drinking water, 30 kg mixed jungle forages (18.4% dry matter and 10.2% crude protein) and 4 kg concentrates (87.1% dry matter and 14.5% crude protein) fortified with mineral mixture and salt. Semen was collected from the animals through rectal massage method. Oxytocin (5 IU, intra muscular) was injected just prior to rectal palpation. Briefly, seminal vesicles were massaged centrally and backwardly

M. Karunakaran et al. / Animal Reproduction Science 101 (2007) 257–264

259

for 5 min followed by the gentle milking of ampullae one by one for 3–5 min, which resulted into erection and ejaculation. During collection, the initial transparent secretions were discarded and neat semen drops were collected in a graduated test tube with the help of a funnel. During the study, all the experimental protocols met the Institute Animal Care and Use Committee regulations. 2.2. Evaluation, dilution and preservation of semen samples A total of 50 ejaculates were collected from the experimental animals over 3 months. Immediately after collection, the samples were kept in a water bath at 37 ◦ C and evaluated for volume, colour, consistency, mass activity and pH. Fresh semen samples were subjected to the determination of mass activity using a 5+ scale (0+ to 5+; 0+ = no motility and 5+ = vigorous motility in wave like pattern) by analyzing four to five fields of view of neat semen drop placed on a prewarmed slide (37 ◦ C) by two persons using bright-field optics (Dewinter binocular microscope; magnification 100×). After the preliminary evaluations, samples were subjected to the initial dilution with pre-warmed (37 ◦ C) 1 ml of tris–egg yolk buffer (Tris–hydroxymethyl aminomethane 3.028% (w/v), sodium citrate 1.655% (w/v), fructose 1.250% (w/v) and egg yolk 20% (v/v); 100,000 IU Penicillium G sodium salt and 100 mg dihydrostreptomycin were added in 100 ml of buffer). The partially diluted samples were then brought to the laboratory in an insulated flask containing warm water (37 ◦ C) for further processing. Spermatozoa concentration was determined in partially diluted semen samples through haemocytometer method (Tomar, 1997) using bright-field optics (Nikon, Eclipse 80i; magnification 400×). Spermatozoa concentration in fresh semen samples was calculated by multiplying the dilution factor of initial dilution with spermatozoa concentration in partially diluted samples. Out of the 50 ejaculates, 20 ejaculates were discarded because of poor quality (mass activity 2+ or less). The remaining 30 ejaculates of good quality (mass activity 3+ or more) were ultimately preserved. The data that presented in the results section are the averages of these 30 samples. Following the determination of spermatozoa concentration, final dilution of the semen samples were done with pre-warmed (37 ◦ C) tris–egg yolk buffer in such a way that after dilution a volume of 1 ml diluted semen contained 25 × 106 spermatozoa. Finally the diluted samples were stored at 4 ◦ C for 72 h. Progressive motility, live spermatozoa count and abnormal spermatozoa count were determined in diluted semen samples at 0 (immediately after final dilution and before storage at 4 ◦ C), 12, 24, 36, 48, 60 and 72 h of storage. The percentage of progressively motile spermatozoa was subjectively determined by two persons to the nearest of 10% by analysing four to five fields of view of sample placed on a pre-warmed slide (37 ◦ C) under pre-warmed cover slip (37 ◦ C) using bright-field optics (Nikon, Eclipse 80i; magnification 400×). Before the determination of progressive motility, the stored samples were warmed in a water bath at 37 ◦ C for 5 min. The count of live spermatozoa was determined using eosine–nigrosine stain (5% (w/v) nigrosine water soluble, 0.6% (w/v) eosine yellow water soluble and 3% sodium citrate dihydrate; filtered and pH adjusted to 7.0 by adding few drops of 0.1 M NaH2 PO4 or 0.1 M Na2 HPO4 ) according to a previously described method (Robeck and O’Brien, 2004) using bright-field optics (Nikon, Eclipse 80i; magnification 1000×). Spermatozoa (eosin–nigrosin stained; 200 per sample) were also evaluated under bright-field optics (Nikon, Eclipse 80i; magnification 1000×) for gross morphological abnormalities. The total morphological abnormality was determined by adding up the proportion of head (abnormal shape; bicephalic; missing; abaxial), midpiece (cytoplasmic droplets; degenerated) and tail (bent; coiled; double) abnormalities.

260

M. Karunakaran et al. / Animal Reproduction Science 101 (2007) 257–264

2.3. Statistical analysis All statistical analyses were performed using SPSS 10.0.1 software package (SPSS, 1999). To study the variations in different recorded parameters, the data were analyzed by means of general linear model. The model included different h of storage and bull as source of variation. All analyses were performed after arcsine transformation of results expressed as percentages. All pair wise multiple-comparison procedures between means were conducted using Student–Newman–Keuls (SNK) test. The data are presented as mean ± S.E. 3. Results The colour, consistency and mass activity of fresh semen samples were found to be creamy white, medium and 3+ to 4+, respectively. The average volume (ml), pH and spermatozoa concentration (106 ml−1 ) of mithun semen were found to be 0.6 ± 0.01, 6.8 ± 0.03 and 425 ± 48, respectively. The variations in progressive motility, live spermatozoa count and total morphological abnormalities at different h of storage are depicted in Figs. 1–3, respectively. Progressive motility (%), live spermatozoa count (%) and total morphological abnormalities (%) were found to be 75.3 ± 3.5, 80.6 ± 4.1 and 5.7 ± 0.2, respectively, at 0 h. Progressive motility and live spermatozoa count decreased significantly (P < 0.01) over the time of storage and were found to be less than 30% after 48 h of storage. Whereas, the total spermatozoa abnormalities increased significantly (P < 0.01) over the time of storage and the highest value was recorded at 72 h. The variations in head, midpiece and tail abnormalities that contributed to the total spermatozoa abnormality at different hours of storage are presented in Table 1. The head (P < 0.05), midpiece (P < 0.05) and tail abnormalities (P < 0.01) increased gradually over the time of storage. Head abnormalities increased significantly above the base values after 36 h of storage and the abnormalities of midpiece and tail increased significantly above the base values after 48 h of storage. However, the maximum variation was observed in tail abnormalities as the time of storage progressed. It was observed that progressive motility, live spermatozoa count and morphological abnormalities did not vary significantly among the bulls.

Fig. 1. Progressive motility (mean ± S.E.) of mithun spermatozoa evaluated at 0, 12, 24, 36, 48, 60 and 72 h of in vitro storage at 4 ◦ C; 0 h indicates the evaluation just before storage; a–d on error bar indicates a significant variation at P < 0.01.

Parameters

Head abnormalities (%) Midpiece abnormalities (%) Tail abnormalities (%)

Time of storage (h)

P

0

12

24

36

48

60

72

1.16 ± 0.01 a 1.21 ± 0.01 a 3.32 ± 0.35 a

1.33 ± 0.03 a 1.35 ± 0.02 a 3.48 ± 0.83 a

1.65 ± 0.08 a 1.62 ± 0.05 a 5.35 ± 0.71 a

2.33 ± 0.16 b 2.12 ± 0.13 a 7.47 ± 1.83 a

2.82 ± 0.25 b 2.32 ± 0.21 b 15.18 ± 3.27 b

3.91 ± 0.29 c 3.32 ± 0.67 c 19.08 ± 3.42 b

4.33 ± 0.35 c 3.91 ± 0.82 d 21.68 ± 4.19 c

Letters a–c: values within row differ significantly; P indicates probability value.

0.02 0.03 0.00

M. Karunakaran et al. / Animal Reproduction Science 101 (2007) 257–264

Table 1 Variations (mean ± S.E.) in head abnormalities (abnormal shape; bicephalic; missing; abaxial), mid piece abnormalities (cytoplasmic droplets; degenerated) and tail abnormalities (bent; coiled; double) of mithun spermatozoa evaluated at 0, 12, 24, 36, 48, 60 and 72 h of in vitro storage at 4 ◦ C; 0 h indicates the evaluation just before storage

261

262

M. Karunakaran et al. / Animal Reproduction Science 101 (2007) 257–264

Fig. 2. Live count (mean ± S.E.) of mithun spermatozoa evaluated at 0, 12, 24, 36, 48, 60 and 72 h of in vitro storage at 4 ◦ C; 0 h indicates the evaluation just before storage; a–c on error bar indicates a significant variation at P < 0.01.

Fig. 3. Total morphological abnormalities (mean ± S.E.; determined by adding up the abnormalities of head, midpiece and tail) of mithun spermatozoa evaluated at 0, 12, 24, 36, 48, 60 and 72 h of in vitro storage at 4 ◦ C; 0 h indicates the evaluation just before storage; a–c on error bar indicates a significant variation at P < 0.01.

4. Discussion To the best of our knowledge the present study is the first report on in vitro preservation of mithun semen. The results indicate that it is possible to preserve mithun spermatozoa at refrigeration temperature using tris–egg yolk diluents. The objective of any semen preservation protocol is to maintain the viability of spermatozoa over an extended period of time. This can be achieved by reducing the metabolism of sperm cell by lowering temperature and through the addition of viability-lengthening diluents (Paulenz et al., 2000). Previous report indicates that gaur semen retains its fertilization capability following the cryopreservation in tris–egg yolk–glycerol extender (Hopkins et al., 1988). It is also established that tris–egg yolk based diluents can be used efficiently to preserve cattle semen and previous studies indicate that semen retains more than 30% progressive motility until 72–144 h of preservation at refrigeration temperature

M. Karunakaran et al. / Animal Reproduction Science 101 (2007) 257–264

263

(Vyas et al., 1991, 1992; Verberckmoes et al., 2004). As mithun is taxonomically related to gaur and cattle, we hypothesized that tris–egg yolk diluent might be suitable to preserve mithun semen at 4 ◦ C. The qualities of fresh semen samples that preserved during the present study were comparatively better than the previous study except spermatozoa concentration (Bhattacharya et al., 2005). Comparatively higher spermatozoa concentration (106 /ml) is reported in the previous report (508 ± 66 versus 425 ± 48). Our results indicated that when mithun semen was preserved at 4 ◦ C, there was a steady decline in progressive motility and live spermatozoa count over the time of storage. Progressive motility and live spermatozoa count were found to be more than 30% and 40%, respectively, until 36 h of storage. Whereas, approximately 10% progressive motility and live spermatozoa count were observed after 72 h of storage. It is advised that cattle semen stored at refrigeration temperature in tris–egg yolk based diluents should be used within 3 days (Verberckmoes et al., 2004). Previous reports indicate that cattle semen retains more than 30% progressive motility until 72 h of preservation, when stored in tris–egg yolk–glycerol diluents at 4–6 ◦ C (Vyas et al., 1991, 1992). However, progressive motility considerably improves, when cattle semen is preserved in tris–egg yolk diluents without glycerol and 30–40% motility is observed even after 144 h of storage at 5 ◦ C (Verberckmoes et al., 2004). In the present investigation, glycerol was not added in the diluent, because glycerol mainly acts as a cryoprotectant and may not be required to preserve semen at refrigeration temperature. Moreover, glycerol induces acrosomal damage, cytoskeletal protein–membrane disruption and alteration in membrane fluidity and metabolic reactions (Holt, 2000). During the study, in one sample, nearly 60% progressive motility was observed even after 72 h of storage, which is not reflected in the given average data. A possible explanation for the observed result is that the animal was probably in a different reproductive state, when sample was collected (Schiewe et al., 1991; Bissett and Bernard, 2005). It was observed that percentage of morphologically abnormal spermatozoa increased steadily over the time of storage. Head, midpiece and tail abnormalities were found to be highest after 72 h of storage. Previous reports in cattle and buffalo also indicate that these morphological abnormalities increase significantly in cryopreserved semen (Bhavsar et al., 1990; Vyas et al., 1992). Our investigation revealed that when mithun semen was preserved in tris–egg yolk diluent at 4 ◦ C, progressive motility and live spermatozoa count remained above 30% and 40%, respectively, until 36 h of storage. Simultaneously the percentages of morphologically abnormal spermatozoa were significantly lower until 36 h of storage. Based on the laboratory trials, the criterion for survival of cryopreserved gaur spermatozoa has been defined as a minimum post-thaw motility of 30% with at least 70% normal morphology (Hopkins et al., 1988). It is also reported that cryopreserved gaur spermatozoa with 30–45% motility are able to establish pregnancy through AI in cow (Sukwongs et al., 1998). Assuming that this level of motility is applicable in mithun, our study revealed that mithun spermatozoa could retain fertilizing ability until 36 h of in vitro storage at 4 ◦ C. However, the results of the present study indicated that mithun semen was probably more susceptible to cold storage and the effective storage period was found to be much shorter compared to the semen of Bos taurus. In conclusion, the tris–egg yolk diluent can be used to preserve mithun semen at refrigeration temperature. An acceptable level of progressive motility, live spermatozoa count and morphologically normal spermatozoa were observed until 36 h of storage, when mithun semen was preserved in vitro at 4 ◦ C using tris–egg yolk diluent. However, further efforts are needed to design suitable diluents to extend the preservation time of mithun spermatozoa and to establish successful pregnancies through AI using the preserved semen.

264

M. Karunakaran et al. / Animal Reproduction Science 101 (2007) 257–264

Acknowledgement We are thankful to Dr. Nikhil Nath for providing the technical assistance during the study. References Bhattacharya, H.K., Goswami, B.K., Bujarbaruah, K.M., Deka, B.C., Baishya, N., Sarma, B.C., 2005. Characteristics of semen collected by massage method in mithun (Bos frontalis) bulls. Ind. J. Anim. Sci. 75, 1168–1169. Bhavsar, B.K., Dhami, A.J., Kodagali, S.B., 1990. Abnormal sperm content in mehsana buffalo bulls semen with regard to freezability, seasonality and fertility. Ind. Vet. J. 67, 233–237. Bissett, C., Bernard, R.T.F., 2005. The effect of prolonged cold storage of eland (Taurotragus oryx) cauda epididymides on the spermatozoa: possible implications for the conservation of biodiversity. Theriogenology 63, 1592–1604. Dee Leeuw, F.E., Dee Leeuw, A.M., Dean Daas, J.H.G., Colenbrander, B., Verkeij, A.J., 1993. Effect of various cryoprotective agents and membrane stabilizing compounds on bull sperm membrane integrity after cooling and freezing. Cryoiology 30, 32–44. Godfrey, R.W., Lunstra, D.D., French, J.A., Schwartz, J., Armstrong, D.L., Simmons, L.G., 1991. Estrous synchronization in the Gaur (Bos gaurus): behavior and fertility to artificial insemination after prostaglandin treatment. Zoo Biol. 10, 35–41. Hammer, C.J., Tyler, H.D., Loskutoff, N.M., Armstrong, D.L., Funk, D.J., Lindsey, B.R., Simmons, L.G., 2001. Compromised development of calves (Bos gaurus) derived from in vitro-generated embryos and transferred interspecifically into domestic cattle (Bos taurus). Theriogenology 55, 1447–1455. Holt, W.V., 2000. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 53, 47–58. Hopkins, S.M., Armstrong, D.L., Hummel, K.C., Junior, S.M., 1988. Successful cryopreservation of gaur (Bos gaurus) epididymal spermatozoa. J. Zoo Anim. Med. 19, 195–201. Johnston, L.A., Parrish, J.J., Monson, R., Leibfried-Rutledge, L., Susko-Parrish, J.L., Northey, D.L., Rutledge, J.J., Simmons, L.G., 1994. Oocyte maturation, fertilization and embryo development in vitro and in vivo in the gaur (Bos gaurus). J. Reprod. Fertil. 100, 131–136. Mondal, S.K., Pal, D.T., 1999. Mithun: historical perspective. Asian Agri-Hist. 3, 245–260. Paulenz, H., Kommisrud, E., Hofmo, P.O., 2000. Effect of long-term storage at different temperatures on the quality of liquid boar semen. Reprod. Dom. Anim. 35, 83–87. Robeck, T.R., O’Brien, J.K., 2004. Effect of cryopreservation methods and precryopreservation storage on bottlenose dolphin (Tursiops truncatus) spermatozoa. Biol. Reprod. 70, 1340–1348. Schiewe, M.C., Bush, M., de Vos, V., Brown, J.L., Wildt, D.E., 1991. Semen characteristics, sperm freezing and endocrine profiles in free-living wildebeest (Connochaetes taurinus) and greater kudu (Tragelaphus strepticeros). J. Zoo. Wildlife Med. 22, 58–72. Simoons, F.J., 1984. Gayal or mithun. In: Manson, I.L. (Ed.), Evolution of Domesticated Animals. Longman, London, pp. 34–36. SPSS, 1999. SPSS® User’s Guide Release 10.0.1. SPSS Inc., USA. Sukwongs, Y., Kamolnorranath, S., Lohachit, C., Apimeteetumrong, M., Yiengvisavakul, V., Wolfe, B., 1998. Successful cryopreservation of gaur (Bibos gaurus) and banteng (Bibos javanicus) spermatozoa and pregnancies after artificial insemination with frozen semen. In: Proceedings, 36th Veterinary Conference. Kasetsart University, Bangkok, p. 8. Tomar, N.S., 1997. Artificial Insemination and Reproduction of Cattle and Buffalos. Sarojprakashan, Allahabad, India. Verberckmoes, S., Van Soom, A., Dewulf, J., De Pauw, I., De Kruif, A., 2004. Storage of fresh bovine semen in a diluent based on the ionic composition of cauda epididymal plasma. Reprod. Dom. Anim. 39, 410–416. Vyas, S., Dhami, A.J., Mohan, G., Sahani, K.L., 1991. Effect of sephadex and glass-wool column filtration on the quality and storage (at 5 ◦ C) of crossbred bull semen. Ind. J. Anim. Sci. 61, 702–704. Vyas, S., Mohan, G., Dhami, A.J., Sahani, K.L., 1992. Studies on the norms and correlations of initial and post-thaw seminal attributes of triple crossbred bulls. Int. J. Anim. Sci. 7, 73–76. Watson, P.F., 1981. The effects of cold shock on sperm cell membranes. In: Morris, G.J., Clarke, A. (Eds.), Effects of Low Temperature on Biological Membranes. Academic Press, London, pp. 189–218.