Effect of Three Days Storage of Coated Spermatozoa at Cooling and Adding Seminal Plasma on Ram Fertility

Document Type : Physiology

Authors

• Alireza Vaferi ¹
• Mohammad Roostaei-Ali Mehr ¹
• Navid Ghavi Hossein-Zadeh ¹
• feridoon talebi ²

¹ Department of Animal Science, Faculty of Agricultural Sciences, University of Guilan, Guilan, Iran

² Agricultural Jihad Organization of Gilan Province, Gilan, Iran

10.22067/ijasr.v8i1.36450

Abstract

Introduction Artificial insemination (AI) has only been used as a supplement to natural mating. AI, when used in conjunction with accurate progeny testing schemes, can substantially increase the rate of genetic progress compared with that of natural service. Moreover, the use of AI causes the limitation of the transmitted diseases. Cervical insemination with frozen-thawed ram semen has not been widely adopted, probably because of the relative poor fertility obtained. Thus using fresh and diluted semen is only approach for performing AI.
AI is currently limited by the poor fertility achieved after cervical insemination with the storage of liquid semen at sub-ambient temperature. The success of this procedure in sheep is restricted by the short length of time that ram sperm can be stored in a liquid state. Moreover, the effect of cooling on sperm differs depending on species. It is also well known that ram spermatozoa are more sensitive to cold-shock stress than those of other species.
Seminal plasma, as physiological secretion, is a complex mixture of secretions originating from testis, epididymis and accessory sex glands which is mixed with epididymal sperm at ejaculation; it serves as the carrier of sperm to the female genital tract. This mixture contains numerous factors such as organic and nonorganic material which play an important role in the final maturation of the spermatozoa through hormonal, enzymatic and surface-modifying events. During natural mating, a mechanism may be activated to separate spermatozoa from seminal plasma. After being ejaculated into the vagina, sperm swim through cervical mucus and enter the uterus within minutes (>30 min); cervical mucus acts as a barrier for seminal plasma. In the artificial insemination industry, seminal plasma with all the useful and harmful components is not removed from semen and is in contact with sperm throughout cooling, freezing and storage.
On the other hand, it was demonstrated that the auto-destructive activity of seminal plasma was decreased which may be reduced by coating spermatozoa for less than 5 min during collection with the commercial diluent supplemented with egg yolk. The detrimental effect of lipid efflux induced by seminal plasma may be abolished by decreasing the time of the contact between seminal plasma and sperm.
The objective of this study was to determine whether coating method, as a collection method, can improve fertility of ram spermatozoa after 72 h storage.
Materials and Methods Experiment was conducted to evaluate the effect of seminal plasma on coated spermatozoa fertility by using 111 ewes, aged between 1 and 3 years. Semen from four mature, healthy and fertile Thaleshi rams, aged between 2 and 5 years, were used for AI. The animals were housed at the Faculty of Agricultural Sciences, Education Research and Practice Farm, University of Guilan, South of Rasht (it is located at 37° 12´ North latitude and 49° 39´ East longitude) and fed daily with alfalfa hay and 0.5 kg of concentrate, and provided salt lick and water ad libitum. Semen was collected throughout the breeding season (August, 2011) by using an artificial vagina. Ejaculates from each ram were collected in a tube containing 5 ml of coating medium (269 mM Tris (Hydroxymethy1) aminomethane, 52 mM D-Fructose, 89 mM Citric Acid, 2000 IU/ml penicillin G and 0.4 mg/ml streptomycin pH=7.0) at72 h before insemination. Two or three consecutive ejaculates fromeach ram were collected. The ejaculates were placed in a water bath (35○C) immediately after collection. Semen quality was assessed, and to be accepted as a donor, and the ejaculation of each ram ejaculation had to fulfill the following demands concerning semen quality: volume ≥ 0.5 ml, macroscopic good visual mass activity (sperm motility ≥ 75%), sperm concentration ≥ 3 × 109⁄ml and normal sperm morphology ≥ 90%. Coated ejaculates were centrifuged for 10 min at 700 × g at room temperature and the supernatant was removed. The pellets were diluted by Tris-glucose up to 800 × 106 sperm/mL then they were split into three parts (E-S+, E-S-and E+S-) and incubated at 5 C. After 68 h‚ samples were centrifuged by 700 × g 10 min at 5 °C. In E-S+, supernatant was removed and added 10% crude seminal plasma. In E-S-‚ supernatant was removed and added Tris-glucose. In E+S-‚ pellet was mixed with supernatant. Samples were packaged into straws‚ incubated at 5 °C for 4 h and inseminated 72 h after collection. Ewes were allocated to three groups and inseminated after synchronizing estrus by using CIDER (14 d) and injection hCG (400 IU).
Results and Discussion The results showed that the lambing rate was higher in ewes of second parity (18.91%) than ewes of first parity (5.12%). There was no significant difference between E-S- (24.32%) and E+S- (10.81%) although the percentage of lambing rate was higher about 10 % in E-S- than E+S-. There was no significant difference between E-S+(5.12%) and E+S- on lambing rate. The pair-wise comparison of the lambing rates between the three groups showed significant higher results for E-S- compared with E-S+. Therefore, fertility of coated spermatozoa was not improved by adding 10% crude seminal plasma after three days storage at 5 C.

Keywords

• Coated spermatozoa Fertility
• Seminal plasma
• Semen storage

20.1001.1.20083106.1395.8.1.1.0

---

References

1- Aboagla, E. M., and T. Terada. 2004. Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa. Theriogenology, 62 (6): 1160-1172.

2- Anel, L., M. Kaabi., B. Abroug., M. Alvarez., E. Anel., J. C. Boixo., L. F. de la Fuente., and P. de Paz. 2005. Factors influencing the success of vaginal and laparoscopic artificial insemination in churra ewes: a field assay. Theriogenology, 63 (4):1235-1247.

3- Bergeron, A., M. Crete., Y. Brindle., and P. Manjunath. 2004. Low-density lipoprotein fraction from hen’s EY decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. Biology of Reproduction, 70 (3): 708–717.

4- Bocquier, F., M. Theriez., S. Prache., and A. Brelurut. 1988. Alimentation des ovins. In: Jarrige J, editor. limentation des bovins, ovins et caprins. Ed. 1, INRA, France, 1: 249–80.

5- De Pauw, I. M. C., D. M. Van Soom., S. Verberckmoes., and A. de Kruif. 2003. Effect of sperm coating on survical and penetrating ability of in vitro stored bovine spermatozoa. Theriogenology, 59 (5-6): 1109–1122.

6- Donovan, A., J.P. Hanrahan., E. Kummen., P. Duffy., and M. P. Boland. 2004. Fertility in the editor. limentation des bovins, ovins et caprins. Ed. 1, INRA, France, 1: 249–80.

7- El-Hajj Ghaoui, R., P. C. Thomson., T. Leahy., G. Evans., and W. M. C. Maxwell. 2007. Autologous whole ram seminal plasma and its vesicle-free fraction improve motility characteristics and membrane status but not in vivo fertility of frozen–thawed ram spermatozoa. Reproduction in Domestic Animals, 42 (5): 541–549.

8- Evans, G. 1991. Application of reproductive technology to the Australian livestock industries. Reproduction, fertility and development, 3 (6): 627–650.

9- Gil, J., M. Rodriguez-Irazoqui., L. Söderquist., and H. Rodriguez-Martinez. 2002. Influence of centrifugation or low extension rates prefreezing on the fertility of ram semen after cervical insemination. Theriogenology, 57 (7): 1781–1792.

10- Gomez-Fernandez, J., E. Gomez-Izquierdo., C. Tomas., A. Gonzalez-Bulnes., R. Sanchez-Sanchez., and E. de Mercado. 2012. Inclusion of seminal plasma in sperm cryopreservation of Iberian pig. Animal Reproduction Science, 130 (1-2): 82– 90.

11- Gordo, A. C., P. Rodrigues., M. Kurokawa., T. Jellerette., G. E. Exley., C. Warner., and R. Fissore. 2002. Intracellular calcium oscillations signal apoptosis rather than activation in vitro aged mouse eggs. Biology of Reproduction, 66 (6): 1828-37.

12- Graham, J. K. 1994. Effect of seminal plasma on the motility of the epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process. Theriogenology, 41 (5): 1151–1162.

13- Kaabi, M., M. Alvarez., E. Anel., C. A. Chamorro., J. C. Boixo., P. de Paz., and L. Anel. 2006. Influence of breed and age on morphometry and depth of inseminating catheter penetration in the ewe cervix: a postmortem study. Theriogenology, 66 (8): 1876-1883.

14- Kirkwood, R. N., M. L. Vadnais., and M. Abad. 2008. Practical application of seminal plasma. Theriogenology, 70 (8): 1364–1367.

15- Krzyzosiak, J., P. Molan., and R. Vishwanath. 1999. Measurements of bovine sperm velocities under true anaerobic and aerobic conditions. Animal Reproduction Science, 55 (3-4): 163-73.

16- Leahy, T., J. I. Marti., N. Mendoza., R. Perez-Pe., T. Muiño-Blanco., J. A. Cebrian-Perez., G. Evans., and W. M. Maxwell. 2010. High pre-freezing dilution improves post-thaw function of ram spermatozoa. Animal Reproduction Science, 119 (1-2): 137-146.

17- Love C. C., T. L. Blanchard., D. D. Varner., S. P. Brinsko., J. Voge., S. Bliss., K. Sudderth., S. Teague., and K. LaCaze 2012. Effect of daily semen centrifugation and resuspension on the longevity of equine sperm quality following cooled storage. Theriogenology, 77 (9): 1911–1917.

18- Manjunath, P., V. Nauc., A. Bergeron., and M. Menard. 2002. Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen's egg yolk. Biology of Reproduction, 67 (4): 1250-1258.

19- Maxwell, W. M. C., and L. A. Johnson. 1999. Physiology of spermatozoa at high dilution rates: the influence of seminal plasma. Theriogenology, 52 (8):1353-62.

20- Maxwell, W. M. C., and S. Salamon. 1993. Liquid storage of ram semen—a review. Reproduction, Fertility and Development, 5 (6): 613–38.

21- O’Meara, C. M., A. Donovan., J. P. Hanrahan., P. Duffy., S. Fair., A. C. O. Evans., and P. Lonergan. 2007. Resuspending ram spermatozoa in seminal plasma after cryopreservation does not improve pregnancy rate in cervically inseminated ewes. Theriogenology, 67 (7): 1262–1268.

22- Paulenz, H., T. Adnøy., T. Fossen., and L. Söderquist. 2008. Effect on field fertility of addition of gelatine, different dilution rates and storage times of cooled ram semen after vaginal insemination. Reproduction in Domestic Animals, 45 (4): 706-710.

23- Ritar, A. J., and S. Salamon, 1982. Effect of seminal plasma and its removal and of egg yolk in the diluent on the survival of fresh and frozen thawed spermatozoa of Angor goat. Australian Journal of Biological Sciences, 35 (3): 305–312.

24- Roostaei-Ali Mehr M., and F. Sharafi. 2013. The effect of seminal plasma on the quality of coated ram frozen-thawed spermatozoa. Iranian Journal of Veterinary Research, 14 (4): 305-312.

25- Shackell, G. H., B. Kyle., and R. P. Littlejohn. 1990. Factors influencing the success of a large scale artificial insemination synchronized oestrus. Animal Reproduction Science, 84 (4): 359-368.

26- Verstegen, J. P., K. Onclin., and M. Iguer-Ouada. 2005. Long-term motilityand fertility conservation of chilled canine semen using egg yolk added Tris-glucose extender: in vitro and in vivo studies. Theriogenology, 64 (3): 720 –33.

27- Yanagimachi, R. 1988. Mammalian fertilization. In: Knobil, E., Neil, J. (Eds.). Physiology of Reproduction. USA, New York, pp. 135-85.