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RESEARCH ARTICLE

Relationship between seminal plasma tuberoinfundibular peptide of 39 residues and sperm functional attributes in buffalo (Bubalus bubalis)

Sellappan Selvaraju A D , Lakshminarayana Somashekar A C , Binsila B. Krishnan A , Sivashanmugam Parthipan A C , Guvvala Pushparani A , Arunachalam Arangasamy A , Duraisamy Rajendran B and Janivara Parameswaraiah Ravindra A
+ Author Affiliations
- Author Affiliations

A Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research (ICAR)–National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India.

B Animal Nutrition Division, Indian Council of Agricultural Research (ICAR)–National Institute of Animal Nutrition and Physiology, Adugodi, Bengaluru 560030, India.

C Department of Biochemistry, Jain University, Bengaluru 560001, India.

D Corresponding author. Email: selvarajuars@rediffmail.com

Reproduction, Fertility and Development 28(10) 1622-1630 https://doi.org/10.1071/RD15008
Submitted: 25 September 2014  Accepted: 12 March 2015   Published: 5 May 2015

Abstract

The buffalo seminal plasma protein profile and its relationship with sperm quality have not been studied in detail. Thus, the aim of the present study was to profile buffalo seminal plasma proteins and to assess the relationship between differentially expressed proteins and sperm characteristics. Semen samples (n = 44) were collected from 11 Murrah buffalo bulls (four ejaculates from each animal) and seminal plasma protein profiling was performed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionisation time-of-flight analysis of one of the differentially expressed proteins, namely the 11–12 kDa protein, identified it as tuberoinfundibular peptide of 39 residues (TIP39). Western blot analysis confirmed the presence of TIP39, with TIP39 expression in seminal plasma varying among bulls. Based on TIP39 levels, bulls were classified into two groups, those with high and low protein. The percentages of spermatozoa positive for mitochondrial membrane potential test, chromatin distribution test, synthetic media sperm penetrability test and acrosomal integrity test were significantly (P < 0.05) high in the high protein group. The present study is the first to demonstrate the presence of TIP39 in buffalo seminal plasma and the positive effect of TIP39 on the functional parameters and fertilising ability of spermatozoa.

Additional keywords: fertility, semen quality.


References

Andrabi, S. M. (2009). Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reprod. Domest. Anim. 44, 552–569.
Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXnslKrtLg%3D&md5=1201e158ad58f3b884332085096c72e4CAS | 18954384PubMed |

Arangasamy, A., Singh, L. P., Ahmed, N., Ansari, M. R., and Ram, G. C. (2005). Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa. Anim. Reprod. Sci. 90, 243–254.
Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXht1egtbzJ&md5=e3d8de1d1c8e18d6bf5837ff579ea6d6CAS | 16260100PubMed |

Arimura, A., Somogyvari-Vigh, A., Miyata, A., Mizuno, K., Coy, D. H., and Kitada, C. (1991). Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testis. Endocrinology 129, 2787–2789.
Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXmsVyntbY%3D&md5=e3b00bd33dd3cb8d8e3c14f6e9ab8c33CAS | 1935809PubMed |

Asadpour, R., Alavi-Shoushtari, S. M., Rezaii, S. A., and Ansari, M. H. (2007). SDS-polyacrylamide gel electrophoresis of buffalo bulls seminal plasma proteins and their relation with semen freezability. Anim. Reprod. Sci. 102, 308–313.
SDS-polyacrylamide gel electrophoresis of buffalo bulls seminal plasma proteins and their relation with semen freezability.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtFKiurjN&md5=69ae78ad375c28cda8f117607c2ec6feCAS | 17433580PubMed |

Aurich, J. E., Kuhne, A., Hoppe, H., and Aurich, C. (1996). Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation. Theriogenology 46, 791–797.
Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD28zgtVKqtQ%3D%3D&md5=b39d3804cd7c458742fb89d67cf7f452CAS | 16727943PubMed |

Barrios, B., Perez-Pe, R., Gallego, M., Tato, A., Osada, J., Muino-Blanco, T., and Cebrian-Perez, J. A. (2000). Seminal plasma proteins revert the cold-shock damage on ram sperm membrane. Biol. Reprod. 63, 1531–1537.
Seminal plasma proteins revert the cold-shock damage on ram sperm membrane.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXnslCrtrs%3D&md5=f9fd636233e2721749f77d78721dbd1fCAS | 11058562PubMed |

Barrios, B., Fernandez-Juan, M., Muino-Blanco, T., and Cebrian-Perez, J. A. (2005). Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold shock. J. Androl. 26, 539–549.
Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold shock.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXmtFKgurc%3D&md5=558d0e60d7094d064ea6794839e398edCAS | 15955894PubMed |

Brandon, C. I., Heusner, G. L., Caudle, A. B., and Fayrer-Hosken, R. A. (1999). Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility. Theriogenology 52, 863–873.
Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXns1OhtbY%3D&md5=87dfe5b4dd4a2f0f08603c9c29358659CAS | 10735126PubMed |

Dahl, M., Perin, T. L., Djurisic, S., Rasmussen, M., Ohlsson, J., Buus, S., Lindhard, A., and Hviid, T. V. F. (2014). Soluble human leukocyte antigen-G in seminal plasma is associated with HLA-G genotype: possible implications for fertility success. Am. J. Reprod. Immunol. 72, 89–105.
Soluble human leukocyte antigen-G in seminal plasma is associated with HLA-G genotype: possible implications for fertility success.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXpslKgs7Y%3D&md5=e0acca674daa775045f120991676286aCAS | 24735563PubMed |

de Souza, F. F., Barreto, C. S., and Lopes, M. D. (2007). Characteristics of seminal plasma proteins and their correlation with canine semen analysis. Theriogenology 68, 100–106.
Characteristics of seminal plasma proteins and their correlation with canine semen analysis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXmtVGht7o%3D&md5=d1d39eff3c38229ac74450109c8797d4CAS | 17512978PubMed |

Della Penna, K., Kinose, F., Sun, H., Koblan, K. S., and Wang, H. (2003). Tuberoinfundibular peptide of 39 residues (TIP39): molecular structure and activity for parathyroid hormone 2 receptor. Neuropharmacology 44, 141–153.
Tuberoinfundibular peptide of 39 residues (TIP39): molecular structure and activity for parathyroid hormone 2 receptor.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXmsVelsA%3D%3D&md5=cee6e3271d2e34a7b18d97c443f1796eCAS | 12559132PubMed |

Dobolyi, A., Palkovits, M., and Usdin, T. B. (2010). The TIP39–PTH2 receptor system: unique peptidergic cell groups in the brainstem and their interactions with central regulatory mechanisms. Prog. Neurobiol. 90, 29–59.
The TIP39–PTH2 receptor system: unique peptidergic cell groups in the brainstem and their interactions with central regulatory mechanisms.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsFyntL7N&md5=338f53f4fe5691cb8d4cb45319452201CAS | 19857544PubMed |

Drabovich, A. P., Saraon, P., Jarvi, K., and Diamandis, E. P. (2014). Seminal plasma as a diagnostic fluid for male reproductive system disorders. Nat. Rev. Urol. 11, 278–288.
Seminal plasma as a diagnostic fluid for male reproductive system disorders.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXmtVShs78%3D&md5=d9f8ba9e006957f43223c7408fb37992CAS | 24709963PubMed |

Drost, M. (2007). Bubaline versus bovine reproduction. Theriogenology 68, 447–449.
Bubaline versus bovine reproduction.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2szpsl2jtA%3D%3D&md5=20c919dda60c6fbe3ed32d47bb3219d8CAS | 17482253PubMed |

Gerena, R. L., Irikura, D., Urade, Y., Eguchi, N., Chapman, D. A., and Killian, G. J. (1998). Identification of a fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase. Biol. Reprod. 58, 826–833.
Identification of a fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXhtlansLo%3D&md5=25ab70b0aab957dee8e3b3b1bab5749bCAS | 9510973PubMed |

Gromova, I., and Celis, J. E. (2006). Protein detection in gels by silver staining: a procedure compatible with mass spectrometry. In Cell Biology a Laboratory Handbook. (Eds J. E. Celis, N. Carter, T. Hunter, K. Simons, J. V. Small and D. Shotton) pp. 219–223. (Elsevier Science: New York, NY).

Harshan, H. M., Singh, L. P., Arangasamy, A., Ansari, M. R., and Kumar, S. (2006). Effect of buffalo seminal plasma heparin binding protein (HBP) on freezability and in vitro fertility of buffalo cauda spermatozoa. Anim. Reprod. Sci. 93, 124–133.
Effect of buffalo seminal plasma heparin binding protein (HBP) on freezability and in vitro fertility of buffalo cauda spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XktFSrsr8%3D&md5=320a82f53d7e19d463e4713130cf686cCAS | 16143473PubMed |

Jobim, M. I., Oberst, E. R., Salbego, C. G., Souza, D. O., Wald, V. B., Tramontina, F., and Mattos, R. C. (2004). Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability. Theriogenology 61, 255–266.
Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXps1Oks7g%3D&md5=401b54506cdad95c35d6e0c3a0d7c764CAS | 14662126PubMed |

Jobim, M. I., Oberst, E. R., Salbego, C. G., Wald, V. B., Horn, A. P., and Mattos, R. C. (2005). BSP A1/A2-like proteins in ram seminal plasma. Theriogenology 63, 2053–2062.
BSP A1/A2-like proteins in ram seminal plasma.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjt1Kjtbk%3D&md5=2997578d7aad653b464ef1d4f90ae5f9CAS | 15823360PubMed |

Juyena, N. S., and Stelletta, C. (2012). Seminal plasma: an essential attribute to spermatozoa. J. Androl. 33, 536–551.
Seminal plasma: an essential attribute to spermatozoa.Crossref | GoogleScholarGoogle Scholar | 22016346PubMed |

Killian, G. J., Chapman, D. A., and Rogowski, L. A. (1993). Fertility-associated proteins in Holstein bull seminal plasma. Biol. Reprod. 49, 1202–1207.
Fertility-associated proteins in Holstein bull seminal plasma.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXivVaitg%3D%3D&md5=7c96999d4a966b88e06985731c27fde0CAS | 8286602PubMed |

Koonjaenak, S., Pongpeng, P., Wirojwuthikul, S., Johannisson, A., Kunavongkrit, A., and Rodriguez-Martinez, H. (2007). Seasonality affects post-thaw plasma membrane intactness and sperm velocities in spermatozoa from Thai AI swamp buffaloes (Bubalus bubalis). Theriogenology 67, 1424–1435.
Seasonality affects post-thaw plasma membrane intactness and sperm velocities in spermatozoa from Thai AI swamp buffaloes (Bubalus bubalis).Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2szlsVGksg%3D%3D&md5=503ba278094d2916796e35b2d1bbe6eeCAS | 17442385PubMed |

Kulkarni, B. A., Fernandes, J. E., Dhande, S. G., and Hegde, U. C. (1996). Characterization of seminal plasma proteins of the Indian buffalo and cattle bulls by SDS-polyacrylamide gel electrophoresis. Buffalo J. 2, 147–156.

Kumar, A., Singh, L. P., Harshan, H. M., and Majumdar, A. C. (2008). Seminal plasma non-heparin binding proteins (NHBP) reduce the cryoinjury to buffalo cauda epididymal spermatozoa induced by heparin binding proteins (HBP). Anim. Reprod. Sci. 104, 220–226.
Seminal plasma non-heparin binding proteins (NHBP) reduce the cryoinjury to buffalo cauda epididymal spermatozoa induced by heparin binding proteins (HBP).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsVOmsLg%3D&md5=1594e9625f054f4eb01dc221461a0a42CAS | 17433582PubMed |

Lacombe, A., Lelievre, V., Roselli, C. E., Muller, J.-M., Waschek, J. A., and Vilain, E. (2007). Lack of vasoactive intestinal peptide reduces testosterone levels and reproductive aging in mouse testis. J. Endocrinol. 194, 153–160.
Lack of vasoactive intestinal peptide reduces testosterone levels and reproductive aging in mouse testis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXptV2ntbo%3D&md5=38e2fd6bd8fb04d3a1caa3a436199701CAS | 17592029PubMed |

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXlsFags7s%3D&md5=8e6d2f0feb3d7e17e4113a8ccd82296cCAS | 5432063PubMed |

Liao, B. Y., and Zhang, J. (2008). Null mutations in human and mouse orthologs frequently result in different phenotypes. Proc. Natl Acad. Sci. USA 105, 6987–6992.
Null mutations in human and mouse orthologs frequently result in different phenotypes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXmt1Git7s%3D&md5=b2c6aa6f753a56b272d6105b0f2088d7CAS | 18458337PubMed |

Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
| 1:CAS:528:DyaG38XhsVyrsw%3D%3D&md5=dcae3d56b563bb1b79df2a95a985d3d0CAS | 14907713PubMed |

Moura, A. A., Koc, H., Chapman, D. A., and Killian, G. J. (2006). Identification of proteins in the accessory sex gland fluid associated with fertility indexes of dairy bulls: a proteomic approach. J. Androl. 27, 201–211.
Identification of proteins in the accessory sex gland fluid associated with fertility indexes of dairy bulls: a proteomic approach.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xit12jtbk%3D&md5=71072e72a827ec98a1fd758e5ca563a3CAS | 16278371PubMed |

Novak, S., Ruiz-Sanchez, A., Dixon, W. T., Foxcroft, G. R., and Dyck, M. K. (2010). Seminal plasma proteins as potential markers of relative fertility in boars. J. Androl. 31, 188–200.
Seminal plasma proteins as potential markers of relative fertility in boars.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXlt1Shtbw%3D&md5=e17eab6b2262fdde95b2ae44935b350bCAS | 19713565PubMed |

O’Leary, S., Jasper, M. J., Robertson, S. A., and Armstrong, D. T. (2006). Seminal plasma regulates ovarian progesterone production, leukocyte recruitment and follicular cell responses in the pig. Reproduction 132, 147–158.
Seminal plasma regulates ovarian progesterone production, leukocyte recruitment and follicular cell responses in the pig.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xot1Ogt74%3D&md5=e8d27c4b58b0c9eb96f1375f0e1fbec1CAS | 16816340PubMed |

Ross, G., Engel, P., Abdallah, Y., Kummer, W., and Schluter, K. D. (2005). Tuberoinfundibular peptide of 39 residues: a new mediator of cardiac function via nitric oxide production in the rat heart. Endocrinology 146, 2221–2228.
Tuberoinfundibular peptide of 39 residues: a new mediator of cardiac function via nitric oxide production in the rat heart.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjslamt70%3D&md5=7ed47ae715cea13f5d476855406aacefCAS | 15677763PubMed |

Selvaraju, S., Ravindra, J. P., Ghosh, J., Gupta, P. S., and Suresh, K. P. (2008). Evaluation of sperm functional attributes in relation to in vitro sperm-zona pellucida binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality. Anim. Reprod. Sci. 106, 311–321.
Evaluation of sperm functional attributes in relation to in vitro sperm-zona pellucida binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1c3ptVSlsQ%3D%3D&md5=e7377eaf292153ff8602865c0956210dCAS | 17576042PubMed |

Selvaraju, S., Reddy, I. J., Nandi, S., Rao, S. B., and Ravindra, J. P. (2009). Influence of IGF-I on buffalo (Bubalus bubalis) spermatozoa motility, membrane integrity, lipid peroxidation and fructose uptake in vitro. Anim. Reprod. Sci. 113, 60–70.
Influence of IGF-I on buffalo (Bubalus bubalis) spermatozoa motility, membrane integrity, lipid peroxidation and fructose uptake in vitro.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXltFelurs%3D&md5=583bea45a3dc05fd81faf1ae502882d0CAS | 18804927PubMed |

Selvaraju, S., Nandi, S., Subramani, T. S., Raghavendra, B. S., Rao, S. B., and Ravindra, J. P. (2010). Improvement in buffalo (Bubalus bubalis) spermatozoa functional parameters and fertility in vitro: effect of insulin-like growth factor-I. Theriogenology 73, 1–10.
Improvement in buffalo (Bubalus bubalis) spermatozoa functional parameters and fertility in vitro: effect of insulin-like growth factor-I.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsVyntLfK&md5=62291ee56aee9a25d1aae9587ad81de4CAS | 19828187PubMed |

Selvaraju, S., Raju, P., Rao, S. B., Raghavendra, S., Nandi, S., Dineshkumar, D., Thayakumar, A., Parthipan, S., and Ravindra, J. P. (2012). Evaluation of maize grain and polyunsaturated fatty acid (PUFA) as energy sources for breeding rams based on hormonal, sperm functional parameters and fertility. Reprod. Fertil. Dev. 24, 669–678.
Evaluation of maize grain and polyunsaturated fatty acid (PUFA) as energy sources for breeding rams based on hormonal, sperm functional parameters and fertility.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XnvFehu78%3D&md5=c76193b8494f4bf38cd72978a5e1b293CAS | 22697117PubMed |

Shivers, B. D., Gorcs, T. J., Gottschall, P. E., and Arimura, A. (1991). Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions. Endocrinology 128, 3055–3065.
Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXkt1altL4%3D&md5=0ef78295ccbbb8c9529200e2b16d301aCAS | 2036976PubMed |

Singh, M., Ghosh, S. K., Prasad, J. K., Kumar, A., Tripathi, R. P., Bhure, S. K., and Srivastava, N. (2014). Seminal PDC-109 protein vis-à-vis cholesterol content and freezability of buffalo spermatozoa. Anim. Reprod. Sci. 144, 22–29.
Seminal PDC-109 protein vis-à-vis cholesterol content and freezability of buffalo spermatozoa.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhvVKitrjO&md5=623d6d26e1d83728780d04b02a403998CAS | 24289867PubMed |

Usdin, T. B., Bonner, T. I., Harta, G., and Mezey, E. (1996). Distribution of parathyroid hormone-2 receptor messenger ribonucleic acid in rat. Endocrinology 137, 4285–4297.
| 1:CAS:528:DyaK28XlvVSmsrw%3D&md5=df5356f8a250a5b89e9f026a165e3840CAS | 8828488PubMed |

Usdin, T. B., Hoare, S. R., Wang, T., Mezey, E., and Kowalak, J. A. (1999). TIP39: a new neuropeptide and PTH2-receptor agonist from hypothalamus. Nat. Neurosci. 2, 941–943.
TIP39: a new neuropeptide and PTH2-receptor agonist from hypothalamus.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXmvFGmu7Y%3D&md5=7b5cdda51b837bfd891981fd38aeacd3CAS | 10526330PubMed |

Usdin, T. B., Paciga, M., Riordan, T., Kuo, J., Parmelee, A., Petukova, G., Camerini-Otero, R. D., and Mezey, E. (2008). Tuberoinfundibular peptide of 39 residues is required for germ cell development. Endocrinology 149, 4292–4300.
Tuberoinfundibular peptide of 39 residues is required for germ cell development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtVKnu77I&md5=c35e88f92fa8e515fd7fb66a8149dd3fCAS | 18483145PubMed |

Watson, P. F. (2000). The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60–61, 481–492.
The causes of reduced fertility with cryopreserved semen.Crossref | GoogleScholarGoogle Scholar | 10844218PubMed |