Study on the in vitro fertilizing potential of mithun (Bos frontalis) semen

Transcription

1 Indian J. Anim. Res., 50 (6) 2016 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Study on the in vitro fertilizing potential of mithun (Bos frontalis) semen P. Perumal* 1, S.K. Srivastava 2, K.K. Baruah 3 and S.K. Ghosh 2 ICAR-National Research Centre on Mithun, Jharnapani, Nagaland , India. Received: Accepted: DOI: /ijar ABSTRACT Semen quality parameters (SQPs) such as motility, viability, total sperm abnormality, integrity of acrosome, plasma membrane and nucleus and vanguard distance travelled by sperm and biochemical profiles such as aspartate amino transaminase (AST), alanine amino transaminase (ALT) and lactate dehydrogenase (LDH) were studied in mithun at post thawed stage. Fifty ejaculates were collected through transrectal massage method from matured bulls and the ejaculates were divided into good and poor quality. The ejaculates were extended with Tris egg yolk citrate glycerol extender and stored in liquid nitrogen. PT seminal, biochemical profiles and zona binding per cent (BP) and binding index (BI) were studied with standard protocols. The results revealed good quality ejaculates has significantly (p<0.05) higher SQPs, BP & BI and has significantly (p<0.05) lower total sperm abnormality, AST, ALT and LDH than in the poor quality ejaculates. PT motility was positively (p<0.05) correlated with SQPs, vanguard distance, BI & BP and negatively correlated (p<0.05) with total sperm abnormality and biochemical profiles. In conclusion, the good quality sperm has higher functional sperm structures and higher intactness of sperm plasma and acrosomal membrane to motile faster in forward direction to reach the site of fertilization and successful fertilization. Key words: Mithun, Post thaw semen, Semen quality parameters. INTRODUCTION Mithun, cattle of mountain is a semi-wild, unique free range bovine species available in north eastern hilly region of India. As per the latest Livestock Census (2007), its population has decreasing especially in Nagaland, Manipur and Mizoram due to lack of suitable breeding strategy which leads to inbreeding in the present population. Greater efforts on suitable and scientific breeding management are needed from all the quarters to preserve and conserve the mithun population in its home tracts. Since, breeding in mithuns is by natural mating with limitations like as cost, poor genetic bulls, disease transmission. Thus, implementation of artificial breeding is utmost essential for improvement of its pedigree. The main objective of the present research on post thaw semen quality parameters is to predict its fertility potential and effectively utilize in artificial breeding programme. SQPs are used widely and varied not only between the bulls, but also between the ejaculates within the same breeding bull and also varied in between the seasons (Raval and Dhami, 2010). SQPs such as post thaw motility, abnormality, intactness of acrosome, plasma membrane and nucleus of spermatozoa are well considered as the important indices of semen quality assessment and were significantly correlated with freezability and/or fertilizing ability of spermatozoa in mammalian *Corresponding author s perumalponraj@gmail.com 1 ICAR-National Research Centre on Mithun, Jharnapani, Nagaland , India. 2 ICAR-Indian Veterinary Research Institute, Izatnagar , Uttar Pradesh, India. 3 ICAR-National Research Centre on Pig, Rani, Guwahati, Assam, India. species (Fiaz et al., 2010). Earlier reports on correlation study revealed that there was highly significant and positive relationship among the SQPs and these SQPs could be applied effectively for practical application in routine semen evaluation to predict preservability, freezability and fertilizing potential of spermatozoa (Bhoite et al., 2005). Perusal of literatures revealed that there is paucity of information on post thaw SQPs of mithun semen. Therefore, the present study was designed with the objective of assess the SQPs of good and poor quality semen at post thaw stage to pursuit future sperm preservation protocols in this precious bovine species. MATERIALS AND METHODS A total of ten healthy mithun bulls of ~ 3 to 5 yr of age, average body weight of 500 kg with good body condition maintained in mithun farm, NRC on Mithun, Jharnapani, Nagaland were selected. They were maintained under uniform feeding, housing and managemental conditions. During the study, all the experimental protocols met the Institutional Animal Care and Use Committee regulations. A total of 50 ejaculates (n=50) were collected through transrectal massage method and were grouped into good and poor quality based on the certain seminal parameters such as mass activity and individual motility of fresh semen. Mass activity +++ and above and individual motility 70 per cent and above

2 910 INDIAN JOURNAL OF ANIMAL RESEARCH were considered as good quality whereas less than +++ and 70 per cent, respectively was considered as poor quality ejaculates. The semen has been extended with standard Tris egg yolk citrate glycerol extender 60 million spermatozoa/ml), frozen with biological freezer and stored in liquid nitrogen. The stored semen straw was thawed at 37 C for 30 seconds. The percentage of post thaw sperm motility (Nikon, Eclipse 80i; magnification 400 ), viability by eosin nigrosin staining and total sperm abnormality, acrosomal integrity by Giemsa staining (Watson, 1975), plasma membrane integrity by hypo-osmotic swelling test (HOST) (Jeyendran et al., 1984), nuclear integrity by Feulgen s staining technique (Barth and Oko, 1989) and vanguard distance travelled by sperm in the bovine cervical mucus (BCMPT) (Matouseket et al., 1989) were determined with standard protocol. Spermatozoa of good and poor quality ejaculates were subjected to heterologous (buffalo oocyte) zona binding assay at post-thaw stage of semen preservation. The zona binding ability of the mithun spermatozoa in terms of BI and BP were assessed as per method used by Fazeli et al. (1993). The leakage of intra cellular enzymes such as AST, ALT and LDH activity were estimated by commercially available kits (Coral Clinical Diagnostic Kits). The results were analysed statistically and expressed as the mean ± S.E.M. The data were analysed with the student t test using the SPSS (version 18.0; SPSS, Chicago, IL). Differences with values of (p<0.05) were considered to be statistically significant after arcsine transformation of percentage data. Correlation among the SQPs and biochemical profiles was established with Pearson s correlation coefficient. Differences at p < 0.05 were considered to be statistically significant. RESULTS AND DISCUSSION In this study, 36% of the good quality semen turned out to be non- freezable and 12% of the poor quality semen gave post thaw motility of 40% or more. Mishra and Tyagi (2006) reported that 5 15% semen stored was discarded due to loss of sperm motility. Singh et al. (2013) and Gebreselassie (2009) reported that good quality semen had higher post thaw motility than poor quality ejaculates. Sharma et al. (1992) reported almost 14.5% reduction in post thaw motility from initial, which could be due to damage Table 1: Mean (±S.E.) post thaw semen quality parameters of good and poor quality mithun semen during the process of cryopreservation. Post thaw motility of spermatozoa of various breeds of cattle bull semen reported by various workers fall between % (Prasad, 1997) to 53% (Mohanty, 1999). Loyi (2008) reported an average post thaw motility of spermatozoa ± 8.28 per cent in crossbred bulls. A probable explanation for the poor post thaw motility and freezability in certain semen samples may be due to massage method of collection and crossbred with other strains of mithun as pure strains of mithun have better SQPs than crossbred mithuns as in cattle (Prasad, 1997; Tyagi et al., 2000). Reduced post-thaw sperm motility and reduced sperm metabolism have been demonstrated in other domestic animal species frozen in extender with egg yolk (Pace and Graham, 1970). In the present study, analysis of post thaw livability revealed that good quality ejaculates have significantly (p<0.05) higher per cent than in poor quality (Singh et al., 2013). Similarly, Gebreselassie (2009) reported post thaw sperm livability was significantly higher in good (51.10 ± 1.86 %) than in poor quality semen (36.92 ± 2.39%). The post thaw livability of spermatozoa was positively correlated (p<0.05) with post thaw motility, acrosomal integrity, plasma membrane, nuclear integrity and BCMPT and negatively correlated (p<0.05) with total sperm abnormality both in good (Table 4) and poor quality semen (Table 5) as reported earlier in cattle (Perumal et al., 2011). The variation in the results could be due to strains and age variation of mithun. This also may be due to strain effect and technical skill of the observer (Tomar et al., 1985). Total morphological abnormality of spermatozoa, in the present study revealed that good quality have significantly (p<0.05) lesser value than in poor quality ejaculates at post thaw stage of semen preservation (Table 1). It was negatively correlated (p<0.05) with post thaw motility, acrosomal integrity, livability, plasma membrane integrity in both the good (Table 4) and poor quality semen (Table 5). In present study, the total morphological sperm abnormality was calculated from head, mid-piece and tail abnormalities. Further, the sperm abnormality may vary due to method of collection, technique employed and temperature shock. Therefore, the difference between the good and poor quality ejaculates may be attributed to these factors (Perumal et al., 2011). Semen Quality Parameters Good quality semen (N=25) Poor quality semen (N=25) Post thaw motility (%) ± 1.96 a ± 1.77 b Livability ± 2.21 a ± 1.97 b Acrosomal integrity (%) ± 2.34 a ± 1.54 b Total sperm abnormality (%) ± 1.54 a ± 1.26 b Plasma membrane integrity (%) ± 2.11 a ± 1.85 b Vanguard distance by CMPT (mm/h) ± 1.26 a ± 1.29 b Nuclear Integrity of spermatozoa (%) ± 1.56 a ± 1.76 b *Means with different superscript within rows differ significantly (p < 0.05) N= Number of ejaculates

3 Volume 50 Issue 6 (2016) 911 Table 2: Mean (±S.E.) post thaw biochemical profiles of good and poor quality mithun semen Biochemical profiles Good quality Poor quality semen (N=25) semen(n=25) AST (µ mole/litre) ± 1.96 a ± 1.77 b ALT (µ mole/litre) ± 2.21 a ± 1.97 b LDH (IU/Litre) ± 2.34 a ± 1.54 b *Means with different superscript within rows differ significantly (p < 0.05) N= Number of ejaculates Detachment of acrosome or loss of acrosomal intactness may result into decreased ATP and loss of intracellular enzymes and proteins from spermatozoa. Owing to loss of acrosomal intactness of spermatozoa may be highly motile but not fertile. Therefore, the acrosomal integrity should always be a major part of assessment of spermatozoa (Kumar et al., 2006). In this study, analysis of parameters revealed that good quality ejaculates have higher number of spermatozoa with intact acrosome than poor quality ejaculates (Table 1). The difference in per cent of post thaw intact acrosome between good and poor quality ejaculates might be due to higher percentage of intact acrosome spermatozoa at fresh stage and often superiority of sperm resistance in good quality in comparison to poor quality. Singh et al. (2013) reported that good quality ejaculates (60.08 ± 1.00 %) had higher post thaw acrosomal integrity of spermatozoa than in poor quality ejaculates (31.92 ± 3.31%). Similarly, Gebreselassie (2009) reported mean post thaw acrosomal integrity of spermatozoa was significantly higher in good (63.17 ± 1.28 %) than in poor quality semen (49.89 ± 1.65 %). Acrosomal integrity was positively correlated with post thaw livability, nuclear, plasma membrane integrity and BCMPT and negatively correlated Table 3: Mean (±S.E.) heterologous zona binding assay of good and poor quality semen at post thaw stage of preservation Parameters Good quality Poor quality semen (N=25) semen (N=25) No of oocyte examined No. of sperm bound oocytes No. of zona bound sperm Binding per cent ± 1.70 a ±1.27 b Binding Index ± 1.38 a ± 1.16 b *Means with different superscript within rows differ significantly (p < 0.05) (p<0.05) with total sperm morphological abnormality in both good (Table 4) and poor quality ejaculates (Table 5) (Gebreselassie, 2009). The acrosomal integrity depends on the factors like age of the bulls, frequency of semen collections, temperature and sexual excitement before collection (Javed et al., 2000). The study of sperm membrane functional status is of particular importance since an intact and functionally active membrane is required for maintenance of sperm motility, metabolism, capacitation, acrosome reaction and successful fertilization (Perumal et al., 2011). The integrity of the sperm plasma membrane, motility and acrosomal integrity of spermatozoa are essential for the fertilizing capacity of spermatozoa. The HOST can assess the functional and physiological aspects of the membrane potentiality. In present study, functional integrity of plasma membrane of spermatozoa was evaluated using HOST, which is a simple quick and inexpensive method. During the HOST, the biochemically active spermatozoa when exposed to hypo-osmotic stress due to influx of water, undergoes swelling and subsequently increase in volume to Table 4: Correlation coefficient among the post thaw semen quality parameters of good quality ejaculates of mithun bulls SQPs Post thaw motility * 0.85* -0.87* 0.81* 0.93* 0.82* Livability * -0.93* 0.85* 0.90* 0.81* Acrosomal integrity * 0.70* 0.85* 0.70* Abnormality * -0.92* -0.82* Plasma membrane integrity * 0.76* Cervical mucus penetration test * Nuclear Integrity 1.00 *Correlation coefficient were significant, p< 0.05 Table 5: Correlation coefficient among the post thaw semen quality parameters of poor quality ejaculates in mithun bulls SQPs Post thaw motility * 0.90* -0.88* 0.92* 0.90* 0.87* Livability * 0.93* 0.92* 0.90* Acrosomal integrity * 0.94* 0.88* 0.92* Abnormality * -0.91* -0.89* Plasma membrane integrity * 0.91* Cervical mucus penetration test * Nuclear Integrity 1.00 * Correlation coefficient were significant, p< 0.05

4 912 INDIAN JOURNAL OF ANIMAL RESEARCH establish equilibrium between the fluid compartment within the spermatozoa and extracellular environment. The osmotic changes induce typical morphological alteration characterized by presence of swollen area in the tail region. These changes have been considered as an indicator of the membrane integrity and normal functional activity of spermatozoa (Jeyendran et al., 1984). In the present study, analysis of this parameter revealed that at post thaw stage of semen preservation, spermatozoa of good quality ejaculates have higher plasma membrane integrity than in poor quality ejaculates (Table 1). Gebreselassie (2009) reported post thaw plasma membrane integrity was significantly higher (p<0.05) in good (40.08 ± 1.46 %) than in poor quality semen (34.17 ± 1.89 %). Post thaw plasma membrane integrity was positively correlated (p<0.05) with post thaw livability, acrosomal integrity, nuclear integrity and BCMPT and negatively correlated (p<0.05) with post thaw total sperm morphological abnormality in good (Table 4) and poor quality ejaculates (Table 5) (Perumal et al., 2011). Routine semen evaluation has certain limitations for comprehensive prediction of fertility of bull semen. The plasma membrane integrity highlights the permeability of sperm membrane to hypo osmotic solution and the projection of higher value is a valid indication of intact membrane and sample with higher value is regarded as potent for establishing pregnancy. In the present study, good quality ejaculates have higher per cent of spermatozoa with nuclear intact than poor quality ejaculates in post thawed stage (Table 1). Further, post thaw nuclear integrity was positively correlated (p<0.05) with post thaw livability, acrosomal, plasma membrane integrity and BCMPT and negatively correlated with post thaw total sperm abnormality in both quality of ejaculates. Perusal of literature did not reveal any study on post thaw nuclear integrity/dna fragmentation in good and poor quality ejaculates in mithun species. So the appropriate comparison could not be substituted by this study. The sperm penetration test is considered as a sperm function test that measures the ability of sperm in the semen to swim up into a column of cervical mucus. The post thaw vanguard distance travelled by sperm was significantly (p<0.05) higher in good than in poor quality (Table 1) ejaculates (Perumal et al., 2011). Post thaw BCMPT was positively correlated (p<0.05) with post thaw motility, livability, acrosomal, nuclear and plasma membrane integrity and negatively correlated (p<0.05) with post thaw total sperm abnormality. Perusal of literature did not reveal any study on post thaw BCMPT in good and poor quality ejaculates in mithun species, but similar study was conducted in cattle that good ejaculates have higher vanguard distance travelled by sperm than by poor quality sperm (Perumal et al., 2011). The difference of distance travelled between good and poor ejaculates would be possible due to the difference in the forward progressive motility and loss of mitochondrial membrane potential. Zona binding assay was carried out to investigate the ability of frozen-thawed mithun spermatozoa to bind with ova from buffaloes (heterologous) (Figure 1, 2). Zona binding assay consisted of determination of BP and BI of post-thawed spermatozoa. The results indicated good quality sperm has positive and significantly higher (p<0.05) post thaw BP and BI than poor quality sperm (Table 3). This is because that the improved level of the post thaw SQPs. Thus it ultimately improved the post thaw BP and BI parameters of sperm in good quality semen. In the present study, the dehydrogenase and transaminase enzyme activities were measured to ascertain the protective action as well as leakage of enzyme during preservation of semen at ultra low temperature. AST and ALT are essential for metabolic processes which provide energy for survival, motility and fertility of spermatozoa and these transaminase activities in semen are good indicators of semen quality because they measure sperm membrane stability (Lopez et al., 1989). Fig 1: Capacitated spermatozoa bound to zona pellucida of a matured oocyte Fig 2: Penetrated oocyte showing first cellular division

5 Volume 50 Issue 6 (2016) 913 Table 6: Correlation coefficient among the post thaw biochemical profiles of good and poor quality ejaculates of mithun Good quality ejaculates Poor quality ejaculates Attributes Attributes AST * 0.71* AST * 0.70* ALT * ALT * LDH 1 LDH 1 * Correlation coefficient were significant, p< 0.05 Thus, increasing the percentage of abnormal spermatozoa in ejaculate causes high concentration of transaminase enzyme in the extra cellular fluid due to sperm membrane damage and ease of leakage of enzymes from spermatozoa (Dogan et al., 2009). Analysis of these enzyme profiles revealed that good quality ejaculates have significantly lower post thawed AST and ALT than poor quality ejaculates (Table 2). Higher activity of AST and ALT in poor quality ejaculates at post thawed seminal plasma clearly indicated that much of the enzyme leaked out in the extracellular fluid following deep freezing due to structural damage, increase cell membrane permeability, destabilize the membrane integrity of acrosome, plasma, mitochondria and flagella of the sperm. Similarly, LDH activity was significantly higher in poor quality ejaculates (Table 2, 6). The reason may be due to damage of sperm plasma membrane or fragile nature of in poor quality sperm than in good quality sperm. CONCLUSION It was concluded that most of the post thaw seminal parameters were significantly higher in good than in poor quality ejaculates indicates good quality sperm has better functional sperm structures like higher intactness of sperm, acrosomal membrane to motile faster in forward direction to reach the site of fertilization and successful fertilization. ACKNOWLEDGMENT The authors were thankful to the Director, ICAR- National Research Centre on Mithun, Jharnapani and the Director, ICAR- Indian Veterinary Research Institute, Izatnagar, Bareilly for their kind help to complete the research work. REFERENCES Barth, A.D. and Oko, R.J. (1989). Preparation of semen for morphological examination. In: Abnormal morphology of bovine spermatozoa, Iowa State University Press, Ames, IA. pp Bhoite, U.Y., Sutar, D.A. and Ulmek, B.R. (2005). Effect of season and period on semen characteristics of two and three breed Gir crosses. Indian J. Anim. Reprod. 26: Dogan, I., Polat, U. and Nur, Z. (2009). Correlations between seminal plasma enzyme activities and semen parameters in seminal fluid of Arabian horses. Iran. J. Vet. Res. 10: Fazeli, A.R., Steenweg, W., Bevers, M.M., de Loos, F.A.M., Van den Broek, J. and Colenbrander, B. (1993). Development of sperm zona pellucida binding assay for bull semen. Vet. Rec. 132: Fiaz, M., Usmani, R.H., Abdullah, M. and Ahmad, T. (2010). Evaluation of semen quality of Holstein Friesian and Jersey bulls maintained under subtropical environment. Pak. Vet. J. 30: Gebreselassie, G.A. (2009). Studies on physico-morphological characteristics and certain enzymes in freezable and nonfreezable semen ejaculates of crossbred bulls. M.V.Sc. Thesis, Indian Veterinary Research Institute (Deemed University), Izatnagar, India. Javed, M.T., Khan, A. and Kauser, R. (2000). Effect of age and season on some semen parameters of Nili-Ravi buffalo bulls. Vet. Arch.70: Jeyendran, R.S., Vander Ven, H. H., Parez-Pelaez, M., Crabo, B.G. and Zaneweld, L.J.D. (1984). Development of an assay to assess the functional integrity of the human membrane and its relationship to other semen characteristics. J. Reprod. Fertil. 70: Kumar, N., Verma, R.P., Singh, L.P., Varshney, V. P. and Dass, S.S. (2006). Effect of different levels and sources of zinc supplementation on quantitative and qualitative semen attributes and serum testosterone level in crossbred cattle (Bos indicus Bos taurus) bulls. Reprod. Nutri. Develop. 46: Livestock Census, India (18 th ). (2007). Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture, Krishi Bhawan, New Delhi, India. López, M.L., Grez, P., Gribbel, I. and Bustos Obregón, E. (1989). Cytochemical and ultrastructural characteristics of the stallion epididymis (Equus cabalus). J. Submicroscopic Cyto. Patho. 21: Loyi, T. (2008). Comparative studies on electrolytes and total seminal plasma protein between freezable and non- freezable crossbred bull semen. M.V.Sc. Thesis, Indian Veterinary Research Institute (Deemed University), Izatnagar, India.

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