Head morphometry and chromatin instability in normal boar spermatozoa and in spermatozoa with cytoplasmic droplets
Thais Schwarz Gaggini, Lays Oliveira Rocha, Elisson Terêncio Souza, Fernanda Marcondes de Rezende, Robson Carlos Antunes, Marcelo Emílio Beletti
Abstract
In boar studs, morphological analyses are used to evaluate sperm quality and thereby categorize ejaculates as either approved or rejected. Normally, morphological characteristics correlate with chromatin disorders, but studies to date have only considered the average of abnormalities; cells were not segregated as normal or abnormal. The aim of this study was to assess whether the presence of cytoplasmic droplets was associated with morphometric characteristics and chromatin instability of spermatozoa heads. Morphological analyses were performed on semen from 11 boars using phase contrast microscopy (200 cells per sample). Normal cells were differentiated from those with cytoplasmic droplets and both types were evaluated separately. Photomicrographs were acquired of normal spermatozoa (Group NOR, N = 1,207) as well as spermatozoa with proximal and distal cytoplasmic droplets (Group DROP, N = 725). Sperm-head morphometry and chromatin structure were evaluated using the toluidine blue technique. Spermatozoa heads in the DROP group were longer (8.37 ± 0.60 × 8.31 ± 0.53; P = 0.025), narrower (4.16 ± 0.21 × 4.19 ± 0.19; P = 0.03), and more symmetric on the sides (0.973 ± 0.012 × 0.971 ± 0.011; P = 0.007) than were spermatozoa heads of the NOR group. The DROP group also had a greater average ellipticity (0.335 ± 0.034 × 0.329 ± 0.031; P = 0.0004), a greater percentage of decondensed chromatin (2.71 ± 3.87 × 2.28 ± 1.38; P < 0.0008), and a greater chromatin heterogeneity (4.66 ± 1.40 × 4.40 ± 1.42; P < 0.0001). A greater frequency of semen collection results in a shorter period of cell maturation and this probably affected the degree of chromatin condensation and the cytoplasmic droplet migration, with concomitant effect on the head morphometry measurements. In conclusion, compared with normal spermatozoa, those with cytoplasmic droplets show altered morphometric characteristics, such as longer and narrower spermatozoa heads. They likewise have greater chromatin instability, resulting in a higher percentage of decondensed chromatin and more heterogeneous chromatin.
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References
Aitken RJ, De Iuliss GN. 2010. On the possible origins of DNA damage in human spermatozoa. Mol Hum Reprod, 16:3-13.
Aitken RJ, Bronson R, Smith TB, De Iuliis GN. 2013. The source and significance of DNA damage in human spermatozoa; a commentary on diagnostic strategies and straw man fallacies, Mol Hum Reprod, 19:475-485.
Arraztoa CC, Miragaya MH, Chaves MG, Trasorras VL, Gambarotta MC, Péndola CH, Neild DM. 2016. Porcine sperm vetrification I: cryoloops method. Androlog, 49. Doi: 10.1111/and.12706.
Beletti ME. 2013. Cromatina espermática: quebrando paradigmas. Rev Bras Reprod Anim, 37:2, 92-96.
Beletti ME, Costa LF. 2003. A systematic approach to multi-species sperm morphometrical characterization. Anal Quant Cytol Histol, 25:97-107.
Beletti ME, Costa LF, Guardieiro MM. 2005. Morphometric features and chromatin condensation abnormalities evaluated by toluidine blue staining in bull spermatozoa. Braz J Morphol Sci, 22:85-90.
Beletti ME, Mello MLS. 2004. Comparison between the toluidine blue stain and the Feulgen reaction for evaluation of rabbit sperm chromatin condensation and their relationship with sperm morphology. Theriogenology, 62:298-402.
Beletti ME, Mello MLS. 1996. Methodological variants contributing to detection of abnormal DNA: protein complexes in bull spermatozoa. Braz J Genet, 19:97-103.
Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, Guérin JF. 2003. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod, 18:1023- 1028.
Boe-Hansen GB, Christensen P, Vibjerg D, Nielsen MBF, Hedeboe AM. 2008. Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility. Theriogenology, 69:728-736.
Boersma AA, Braun J, Stolla R. 1999. Influence of random factors and two different staining procedures on computer-assisted sperm head morphometry in bulls. Reprod Dom Anim, 34:77-82.
Briz MD, Bonet S, Pinart B, Egozcue J, Camps R. 1995. Comparative study of boar sperm coming from the caput, corpus, and cauda regions of the epididymis. J Androl, 16:175-88.
Bungum M. 2012. Sperm DNA integrity assessment: a new tool in diagnosis and treatment of fertility. Obstet Gynecol Int, 2012. Doi.org/10.1155/2012/531042.
Bungum M, Humaidan P, Spano M, Jepson K, Bungum L, Giwercman A. 2004. The predictive value of sperm chromatin assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod, 19:1401-1408.
Cooper TG, Yeung CH. 2003. Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet. Microsc Res Techniq, 61:28-38.
Erenpreiss J, Bars J, Lipatnikova V, Erenpreisa J, Zalkalns J. 2001. Comparative study of citochemical tests for sperm chromatin integrity. J Androl, 22:45-53.
Erenpreiss J, Jepson J, Giwercman A, Tsarev I, Erenpreisa J, Spano M. 2004. Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod, 19:2277- 2282.
Feitsma H, Bergsma R, Leenhouwers JI, Knol EF. 2008. Combining AI and Breeding Databases for Analysing the Relation between Boar Semen and Sow Fertility Variables. Reprod Dom Anim, 43:57-58.
Feitsma H. 2009. Artificial insemination in pigs, research and developments in The Netherlands, a review. Acta Sci Vet, 37:61-71.
Flowers WL. 2004. Detailed description of sperm motility/morphology and causes of abnormalities. In Abstracts of the Midwest Boar Stud Conference II, 2004, St Louis, USA, p.15-22.
Fischer MA, Willis J, Zini A. 2003. Human sperm DNA integrity: correlation with sperm cytoplasmic droplets. Urology, 61:207-2011.
Gaggini TS, Rocha LO, Moura TS, Antunes RC, Beletti ME. 2015. Spermatic morphometry of boars approved and reproved in spermatic morphology test [In Portuguese]. In Abstracts of the XVII Congresso da Associação Brasileira de Veterinários Especialistas em Suínos, 2015, Campinas, Brazil. Campinas: ABRAVES, p.448-450.
Gil MC, García-Herreros M, Aparicio IM, Santos AJ, García-Marín LJ. 2009. Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen. Theriogenology, 71:254-263.
Golan R, Cooper TG., Oschry Y, Oberpenning F, Schulze H, Shochat L, Lewin LM. 1996. Changes in chromatin condensation of human spermatozoa during epididymal transit as determined by flow cytometry. Hum Reprod, 11:1457-1462.
Gomez E, Buckingham DW, Brindle J, Lanzafame F, Irvine DS, Aitken RJ. 1996. Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. J Androl, 17:276-287.
Hancock JL, Hovel GLR. 1959. The collection of boar semen. Vet Rec, 71:664-665.
Hingst O, Blottner S, Franz C. 1995. Chromatin condensation in cat spermatozoa during epididymal transit as studied by aniline blue and acridine orange staining. Andrology, 27:275-279.
Hirai M, Boersma A, Hoeflich A, Wolf E, Foll J, Aumüller TR, Braun J. 2001. Objectively measured sperm motility and sperm head morphometry in boars (Sus scrofa): relation to fertility and seminal plasma growth factors. J Androl, 22:104-10.
Irvine DS, Twigg JP, Gordon E, Fulton N, Milne PA, Aitken RJ. 2000. DNA integrity in human spermatozoa: relationships with semen quality. J Androl, 21:33-44.
Januskauskas A, Johannisson A, RodríguezMartínez H. 2003. Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility. Theriogenology, 60:743-758.
Karabinus DS, Vogler CJ, Saacke RG, Evenson DP. 1997. Chromatin structural changes in sperm after scrotal insulation of Holstein bulls. J Androl, 18:549-555.
Khalil Y. 2004. Binding capacity of bull spermatozoa to oviductal epithelium in vitro and its relationship to sperm chromatin stability, sperm volume regulation and fertility. (thesis). Hanover, Germany: School of Veterinary Medicine Hannover.
Kondracki S, Iwanina M, Wysokińska A, Huszno M. 2012. Comparative analysis of Duroc and Pietrain boar sperm morphology. Acta Vet, 81:195-199 Liu DY, Baker HWG. 1992. Sperm nuclear chromatin normality: relationship with sperm morphology, spermzona pellucida binding, and fertilization rates in vitro.
Fertil Steril, 58:1178-1184. Lucio AC, Alves BG, Alves KA, Martins MC, Braga LS, Miglio L, Alves BG, Silva TH, Jacomini JO, Beletti ME.2016. Selected sperm traits are simultaneously altered after scrotal heat stress and play specific roles in in vitro fertilization and embryonic development. Theriogenology, 86(4):924-933.
Martinez FA. 2005. Studies on the interaction of chromatin-unstable boar sperm with the female reproductive tract. (thesis). Hannover, Germany: University of Veterinary Medicine Hannover.
McPherson FJ, Nielsen SG, Chenoweth PJ. 2014. Semen effects on insemination outcomes in sows. Anim Reprod Sci, 151:28-33.
Naves CS, Beletti ME, Duarte MB, Vieira RC, Diniz EG, Jacomini JO. 2004. Evaluation of equine spermatic chromatin with toluidine blue and acridine orange. Biosci J, 20(3):117-124. Pursel VG, Johnson LA, Rampacek GB. 1972. Acrosome morphology of boar spermatozoa incubated before cold shock. J Anim Sci, 34:278-283.
Rodrigues ACN, Rocha JV, Beletti ME. 2009. Análise computacional da compactação da cromatina de espermatozoides de galo. Arq Bras Med Vet Zootec, 61(6):1302-1307.
Saravia F, Núnez-Martínez I, Morán JM, Soler C, Muriel A, Rodríguez-Martínez H, Penã FJ. 2007. Differences in boar sperm head shape and dimensions recorded by computer-assisted sperm morphometry are not related to chromatin integrity. Theriogenology, 68:196-203.
SAS Institute. 2005. SAS User’s Guide, Release 9.1.3. SAS Institute, Cary, NC.
Spano M, Bonde JP, Hjøllund HI, Kolstad HA, Cordelli E, Leter G. 2000. Sperm chromatin damage impairs human fertility. Fertil Steril, 73(1):43-50.
Tsakmakidis IA, Lymberopoulos AG, Khalifa TA. 2010. Relationship between sperm quality traits and field-fertility of porcine semen. J Vet Med Sci, 11:151- 154.
Volker G. 2004. Untersuchungen zur Verbesserung der In-vitro-Diagnostik von Eberspermien und Ermittlung der Beziehung zum Befruchtungserfolg. [in German]. (Thesis). Hanover, Germany: School of Veterinary Medicine Hannover.
Waberski D, Helms D, Beyerbach M, Weitze KF, Bollwein H, Bluemig P, Willeke H, Acevedo N, Saacke RG. 2002. Sperm chromatin structure in boars used in artificial insemination. Reprod Dom Anim, 37:257.
Waberski D, Meding S, Dirksen G, Weitze KF, Leiding C, Hahn R. 1994. Fertility of long-term–stored boar semen: influence of extender (Androhep and Kiev), storage time and plasma droplets in the semen. Anim Reprod Sci, 36:145-151.
Yoseffi S, Oschry Y, Lewin LM. 1994. Chromatin condensation in hamster sperm: a flow cytometric investigation. Mol Reprod Dev, 37:93-98.
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