Research Article

 

 

Effect of Dilution, Cooling and Freezing on Physical and Biochemical Properties of Semen for Holstein Bull Born in Iraq

 

Souhayla Oneeis Hussain*, Hajer Kadhem Shahad, Kreem Iwaid Al-Badry

Department of Theriogenology, College of Veterinary Medicine, University of Baghdad, Iraq.

 

Editor | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.

Received | September 27, 2016; Accepted | October 27, 2016; Published | November 05, 2016

Correspondence | Souhayla Oneeis Hussain, Department of Theriogenology, College of Veterinary Medicine, University of Baghdad, Iraq; Email: Souhayla96@yahoo.com

Citation | Hussain SO, Shahad HK, Al-Badry KI (2016). Effect of dilution, cooling and freezing on physical and biochemical properties of semen for holstein bull born in Iraq. Adv. Anim. Vet. Sci. 4(11): 575-579.

DOI | http://dx.doi.org/10.14737/journal.aavs/2016/4.11.575.579

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2016 Hussain et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

Introduction

 

Artificial Insemination (AI) has the ability to promote quick and broad improvement in genetic quality and opened a new many aspect in animal breeding process through enhancing the selection activity that become a huge economic importance in improving production characteristics of local animals in a short time interval in comparison with natural mating (Bearden et al., 2004). The AI success basically depends on getting semen of a highly fertility, semen consist of seminal plasma and sperms. Seminal plasma composed of a secretion from the male accessory sex glands and epididymis, which contain many organic and inorganic components that have many effects on semen quality (Foxcroft et al., 2008). study the activity of enzymes such as GOT, GPT, ACP and ALP and proteins in the seminal plasma are essential for metabolic process which provide energy for viability, motility and fertility of spermatozoa in relation to the freezability of semen (Pangawkar et al., 1988), and using this parameter as a valuable tool to evaluate the fertility potential of the male (Akusu et al., 1984; Salaam, 2008). Cryopreservation coupled with artificial insemination made rapid strides in genetic improvement of livestock world over during the last millennium. However, the process of freezing and thawing of semen has deleterious effects on post-thaw semen characteristics and fertility (Mutha et al., 2012). Frozen semen in straws has become the universally accepted unit of storage and transfer of bovine genetics to cattle producers. The main factors assumed to be potentially involved in the damage at spermatozoa DNA during the freezing-thawing process are the osmotic stress and the oxidative stress that occur after thawing (Alvarez and Storey, 1992; Batellier et al., 2000). Longer-term storage of semen is achieved through cryopreservation. It has yet some adverse effect on the spermatozoa manifested as a depression in viability rate, structural integrity, depressed motility and conception rates (Watson, 2000; Batellier et al., 2000; Medeiros et al., 2002). In Iraq several studies proved that the steps of freezing (dilution, cooling, freezing rate and thawing) have an effect on physical properties of semen, but in the view of this and since little information are available concerning various aspect of the biochemical components of the semen of Holstein bulls born and reared in Iraq, the present study will conduct to monitor the changes in the activity of these enzymes during the steps of processing frozen semen.

 

Materials and Methods

 

Bulls

 

Ejaculates were collected weekly by artificial vagina from five Holstein bulls born in Iraq age (4-5 year) and maintained at Artificial Insemination Center-Baghdad, the ejaculates which were collected was consider to be poor ejaculate which have individual motility 45-50% and good more than 50%.

 

Evaluation of Semen Quality

 

Physical Properties: A total of 43 ejaculates were studied during period of study. After collection of semen, the sample was immediately brought to the laboratory and placed in a water bath at (37 to 38°C) and graduated tube used for estimated ejaculate volume , mass and individual motility (Chemineau et al., 1991). Dead and abnormalities percentages were evaluated using Eosin-Nigrosin stain (Bearden et al., 2004).

 

Biochemical Evolution: Immediately after application of few drops of semen needed for various parameters for evaluation of fresh semen. Two ml of semen divided to two equal samples, 1ml centrifuged for 30 minute at 4000rpm at 10 C°to separate seminal plasma (leaking) and checking this is under microscope to insure that it was sperm free, then it was transferred to sterile vials and stored at -20C°for future analysis of enzymes and protein. Biochemical analysis was performed with an automatic analyzer Tossoh System (Hitachi Bothering Mannheim, 912 Automatic Analyzer) using specific kits to Measured: Enzymes such as, Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvic Transaminase (GPT), Alkaline Phosphatase (ALP), Acid Phosphatase (ACP) and Total Protein. At the same time the other 1ml of semen sample was diluted by Tris-egg yolk- fructose (dilution rate 1:10), and freezing in liquid nitrogen after package in mini straws by done steps of freezing (dilution, cooling, thawing) and evaluated the physical and biochemical properties of semen during each steps.

 

Statistical Analysis

 

The Statistical Analysis System according to program of (SAS, 2012) was used to effect of different factors in study parameters. T-Test and Duncan multiple range tests were used to determine the significant differences between means in this study (Duncan, 1955).

 

Results

 

Physical Properties

 

Results of the study depicted in Table 1 proved a significant decrease (p<0. 05) in the percentage of individual motility of both poor and good ejaculate during the different steps of processing frozen semen in comparison with fresh semen. The study also proved to result in Table 1 significant difference (p<0. 05) in the percentage of individual motility between poor and good ejaculate in all steps of processing the frozen semen.

 

Table 1: Affect the steps of freezing on semen characteristics for poor & good ejaculate of Holstein bulls born in Iraq (Mean ± SE)

 

Characters	Quality	Fresh semen	Steps of freezing
			Dilution	Cooling	Freezing
individual motility (%)	Poor	34. 79 ± 2. 98 a	28. 33 ± 2. 34 ab	26. 67 ± 2. 22 b	25. 62 ± 2. 07 b
	Good	59. 06 ± 1. 04 a	53. 75 ± 1. 25 b	52. 50 ± 1. 44 bc	50. 00 ± 1. 29 c
	T-test	7. 63 *	6. 18 *	6. 02 *	5. 57 *
Dead (%)	Poor	22. 12 ± 1. 09 b	25. 21 ± 1. 40 b	29. 54 ± 0. 95 b	38. 50 ± 1. 62 a
	Good	17. 43 ± 1. 23 b	19. 18 ± 1. 23 b	20. 62 ± 1. 68 b	29. 62 ± 1. 15 a
	T-test	3. 38 *	4. 04 *	3. 65 *	4. 47 *
Abnormality (%)	Poor	12. 00 ± 1. 15 b	13. 21 ± 1. 12 b	10. 95 ± 0. 67 b	20. 91 ± 1. 27 a
	Good	7. 44 ± 0. 63 b	8. 81 ± 0. 67 ab	11. 25 ± 1. 32 a	15. 94 ± 0. 83 a
	T-test	3. 06 *	3. 00 *	2. 74 NS	3. 46 *

 

Within row different small letters for each parameter means significant at (p<0. 05); Within column in each parameter (T Test); *: significant (P<0. 05); NS: Non-significant

 

Table 2: Affect the steps of freezing on Enzymes and Total protein in poor & good ejaculate of Holstein bulls born in Iraq (Mean ± SE)

 

Biochemical properties	Quality	Fresh semen	Steps of freezing
			Dilution	Cooling	Freezing
GOT U/L	Poor	867. 58 ± 79. 19 c	1641. 96 ± 78. 20 b	1896. 71 ± 78. 69 bc	6794. 08 ± 94. 1 a
	Good	594. 25 ± 89. 84 c	1572. 56 ± 75. 1 bc	2375. 88 ± 63. 7 b	5748. 19 ±75. 8 a
	T-test	117. 51 *	532. 1NS	362. 13*	201. 024*
GPT U/L	Poor	50. 67 ± 4. 11 d	63. 75 ± 2. 34 c	73. 12 ± 5. 59 b	97. 79 ± 10. 61 a
	Good	40. 93 ± 4. 71 b	46. 00 ± 14. 09 b	47. 87 ± 9. 61 b	67. 31 ± 17. 99 a
	T-test	12. 82*	23. 86*	21. 06 *	39. 63*
ALP KAU/L	Poor	22033. 75 ± 788. 7 b	25338. 71 ±750. 4 b	26892. 0 ±821. 4 b	37345. 58 ±892. 2 a
	Good	12883. 31 ± 904. 9 c	13941. 56 ±746. 1 c	15050. 81 ±889. 1 b	18950. 19 ±537. 1 a
	T-test	6582. 0 *	6627. 4*	4053. 3*	2623. 6*
ACP KAU/L	Poor	1050. 21 ± 188. 68 c	1240. 33 ± 173. 1 c	1811. 17 ± 111. 32 b	2772. 13 ±133. 34 a
	Good	962. 81 ±108. 1 b	1006. 88 ± 112. 97 b	1103. 56 ± 100. 96 b	2073. 25 ± 158. 96 a
	T-test	678. 7 NS	4150. 2*	537. 3*	670. 17*
Total protein gm/L	Poor	69. 29 ± 3. 67 b	189. 87 ± 15. 52 b	455. 91 ± 42. 85 a	586. 04 ± 141. 4 a
	Good	56. 25 ± 4. 64 b	374. 31 ± 85. 01 a	574. 871 ± 6. 29 a	541. 43 ± 106. 4 a
	T-test	11. 91 *	144. 85 *	152. 43NS	393. 14NS

 

Within row different small letters for each parameter significant at (p<0. 05); within column in each parameter (T Test); *: significant (P<0. 05); NS: Non-significant

 

The dead and abnormalities values was depicted in Table 1 proved a significant increase (p<0. 05) for both poor and good ejaculate during the different steps of processing frozen semen, and a significant differences (p< 0. 05) between poor and good ejaculate in percentage of both dead and abnormal sperm had also been recorded.

 

Biochemical Evaluation

 

Values of GOT during different steps of freezing are depicted in Table 2, the result indicate a gradual increase in the value of enzyme during the different steps of processing and highest significant value was observed in frozen semen in comparison with fresh semen, diluted and cooled semen. Regarding the differences in values of GOT between poor and good ejaculates, the study proved high significant value (p< 0. 05) was observed in poor ejaculate in comparison with good ejaculate during all steps of processing the frozen semen. Table 2 also shows that values of GPT during different steps of freezing indicate a gradual increase in the value of enzyme during the different steps of processing and highest significant value was observed in frozen semen in comparison with fresh semen, diluted and cooled , regarding the differences in values of GPT between poor and good ejaculate, the study proved high significant value (p< 0. 05) was observed in poor ejaculate in comparison with good ejaculate during all steps of processing the frozen semen. On the other hand ALP enzyme also showed increase during the different steps of processing and highest significant value was observed in frozen semen in comparison with fresh semen, diluted and cooled (Table 2), regarding the differences in values of ALP between poor and good ejaculate, the study proved high significant value (p< 0. 05) was observed in poor ejaculate in comparison with good ejaculate during all steps of processing the frozen semen. In addition to values of ACP during different steps of freezing are depicted in Table 2, the result indicate a gradual increase in the value of enzyme during the different steps of processing and highest significant value was observed in frozen semen in comparison with fresh semen, diluted and cooled, and ACP enzyme high significant (p< 0. 05) in poor ejaculate in comparison with good ejaculate during all steps of processing the frozen semen. Table 2 depicted values of total protein concentration during different steps of freezing, the result indicate a gradual increase in the value of protein during the different steps of processing and highest significant value was observed in frozen semen in comparison with fresh semen diluted and cooled, and total protein high significant (p< 0. 05) in poor ejaculate in comparison with good ejaculate during all steps of processing the frozen semen.

 

Discussion

 

Physical Properties

 

Significant decrease in individual motility and increase in dead and abnormalities percentage for both poor and good ejaculate during different steps, dilution, cooling and freezing of bull semen, this might be attributed to the fact that lactic acid which produced as an end product of sperm metabolism, resulting in harmful lowering of PH which exerts toxic effect on sperm cell (Ball and Peter, 2004). The considerably reduced values for sperm motility, viability, morphology, and plasma membrane/acrosome integrity observed after cryopreservation of semen over fresh or pre-freeze stage (Chaudhari et al., 2015).

 

Biochemical Evaluation

 

Regarding the investigation of transaminases and phosphatases values and concentration of total protein in the seminal of the Holstein bull during different stage of dilution, cooling and freezing of semen proved that there is gradual significant increase in these parameters during the different steps of frozen semen, and that is true since it has been found by earlier worker (Gowda et al., 2010; Sharma et al., 2013). The above mention parameters in the seminal plasma considered as index of sperm damage during dilution, cooling and freezing leading to increase sperm membrane permeability both in good & poor ejaculate but the effect is on poor ejaculate in comparison with good ejaculate (Pangawkar et al., 1988; Szasz et al., 2000; Zedian et al., 2008; Sharma et al., 2013; Srivastava and Kumar, 2014). It appears that spermatozoa damage during storage may be associated with increase sperm membrane permeability and leakage of intracellular enzymes (El-Harairy et al., 2011). The leakage of enzymes from sperm cell into the seminal plasma was very severe. Compared to fresh samples more than 8 fold increases in enzymatic activities of the GOT and GPT and almost 2 folds of ACP and AKP in the frozen-thawed seminal plasma was observed. This increase in enzymatic activity in the frozen-thawed seminal plasma indicates increased cell membrane permeability and damages to the sperm cells during the process of freezing and thawing.

 

Conclusion

 

Physical properties such as individual motility decrease significantly during dilution, cooling and freezing but percentage of dead and abnormalities and biochemical enzymes and total protein for both poor and good ejaculates increased significant during these steps of freezing.

 

Acknowledgements

 

This study was carried out in the Artificial Insemination Center, Iraq. The authors would like to thank the membersof the AI Center, especially to Dr. Faris Fasil Ibrahim and Dr Kamel Fayath and Mrs. Wafa’a Yeedam Lateeffor their unlimited help.

 

Authors’ Contribution

 

All authors contributed equally in all the efforts for these articles.

 

Conflict Of Interest

 

Authors declare that there is no conflict of interest.

 

References

 

Akusu MO, Agiang EA, Egbunike GN (1984). Ejaculate and seminal plasma characteristics of the west African Dwarf (WAD) buck. 10th Int. Cong. On Anim. Prod. And A. I. Pp. 50.

Alvarez JA, Storey BT (1992). Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a model of sublethal cryodamage to human sperm during cryopreservation. J. Androl. Review. 13: 232– 241.

Ball PJH, Peter AR (2004). Reproduction in cattle. 3rd edition. Blackwell Publishing, Oxford. http://dx.doi.org/10.1002/9780470751091

Batellier F, Vidament M, Fauquant J, Duchamp G, Arnaud J, Yvon MA, Bilodeau JFS, Chatterjee FS, Sirard MA (2000). Level of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol. Reprod. Dev. 55: 282-288. http://dx.doi.org/10.1002/(SICI)1098-2795(200003)55:3<282::AID-MRD6>3.0.CO;2-7

Bearden HJ, Fuquay JW, Willard ST (2004). Applied Animal Reproduction. 6th edition. Pearson Prentice Hall, New Jersey.

Chaudhari DV, Dhami AJ, Hadiya KK, Pate JA (2015). Relative efficacy of egg yolk and soya milk-based extenders for cryopreservation (−196 °C) of buffalo semen. http://dx.doi.org/10.14202/vetworld.2015.239-244

Chemineau P, Cognine Y, Guerin Y, Orgeure P, Valtet JC (1991). Training manual on artificial insemination in sheep and goats. FAO, Animal Production and health. Pp: 83.

Duncan DB (1955). Multiple Rang and Multiple F-test. Biometrics. 11: 4-42. http://dx.doi.org/10.2307/3001478

El-Harairy MA, Laila N, Zeidan AEB, Abd El-Salaam AM, El-Kishk MAM (2011). Quality and fertility of the frozen-thawed bull semen as affected by the different cryoprotectants and glutathione levels. J. Am. Sci. 7(5): 791-801.

Foxcroft, Dyck MK, Ruiz-Sanchez A, Novak S, Dixon WT (2008). Identifying unable semen. Theriogenology. 70: 1324-1336.

Gowda HC, Roychondhury PN, Pareek PK (2010). Transaminase activity of bull semen: Effect of buffers on Extracellular enzymes release. Zentralbl Veterinarmed A. 22: 341-345. http://dx.doi.org/10.1111/j.1439-0442.1975.tb01438.x

Medeiros CM, Forell F, Oliviera ATD, Rodrigues JL (2002). Current status of sperm Cryopreservation: Why Isn’t it Better. Theriogenol. 57: 327-344. http://dx.doi.org/10.1016/S0093-691X(01)00674-4

Mutha MA, Verma PA, Ahmed MB, Pandey AK, Raina R (2012). Effect of anti-oxidant supplementation on post thaw sperm characteristics, cellular enzyme leakage and lipid per oxidation in bull semen. Indi. J. Anim. Reprod. 33(1): 29-32.

Pangawkar GR, Sharma RD, Sharma R (1988). Phosphatase and Transminase activity in the seminal plasma of bulls in relation to freezability of semen. Theriogenol., 29(6): 1393-1399. http://dx.doi.org/10.1016/0093-691X(88)90020-9

Salaam AH (2008). Viability and enzymatic leakage of the cooled camel spermatozoa in relation to different extenders. American Eurasian J. Agric. Environ. Sci. 4: 142-149.

SAS (2012). Statistical Analysis System, User’s Guide. Statistical. Version 9. 1st ed. SAS. Inst. Inc., Cary, NC, USA.

Sharma SS, Sunanda S, Sharma K, Sinha NK (2013). Assessment of Transaminases and effect of freezing rate on theor leakage into seminal plasma of Sirohi bucks. Eur. Urol. 2: 18-22.

Srivastava S, Kumar S (2014). Incorporation of ascorbic acid, caffeine and chloroquine diphosphate in dilutor improves structural and functional status of frozen semen. Open Access Lib. J. 1: 1-12.

Szasz F, Gabor G, Solti L (2000). Comparative study of different methods for dog semen cryopreservation and testing under clinical conditions. Acta Vet. Hungarica. 48: 325-333. http://dx.doi.org/10.1556/AVet.48.2000.3.9

Watson PF (2000). The cause of reduces fertility with cryopreserved semen. Anim. Reprod. Sci. 60: 481-492. http://dx.doi.org/10.1016/S0378-4320(00)00099-3

Zedian AEB, Abou-Aiana RM, Ahmadi AM, Abd El-Aziz NA, Sayah NS, Ammar AH, Abd EL-Salam AH (2008). Viability and enzymatic leakage of the cooled camel spermatozoa in relation to different extenders. American Eurasian J. Agric. Environ. Sci. 4: 142-149.