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Aims of the study
Although there have been a number of studies on the collection of spermatozoa and on aspects of the application of AI techniques in the emu (Malecki et al., 1997; 2008; Sood et al., 2011, 2012), it is clear from the available literature that there is a paucity of basic information regarding sperm development, sperm morphology and the classification of defective sperm in the emu. For AI to be consistently successful, it is important to establish the normal parameters for emu semen and spermatozoa. Detailed descriptions of abnormal forms of spermatozoa in the emu are an essential prerequisite for accurate semen evaluation. This information is currently limited to a brief and superficial report by Malecki et al. (1998b) and the cursory description of the ultrastructure of normal emu sperm by Baccetti et al. (1991). Accordingly, the present study aims to document in detail the process of spermiogenesis in the emu and in particular those aspects which have not previously been addressed in the literature. It further aims to provide detailed descriptions on the morphology of normal as well as abnormal spermatozoa. This approach will potentially provide baseline data of value for the application of AI in the industry and for other farming practices such as the selection of male birds with good semen parameters. The incidence of sperm defects will be determined and, where possible, the origin and formation of the defective sperm will be described. The apparent confusion regarding the terminology currently in use to describe sperm abnormalities in birds will be addressed.
Light microscopy
Semen samples were collected during mid-breeding season from 15 healthy (animals approved for slaughter) and sexually active emus following slaughter at commercial abattoirs. Two groups of birds were sampled. One group (n=5) was sourced from the Rustenburg district in the North West Province, South Africa and slaughtered at The Emu Ranch Abattoir. The second group of birds was from the Grahamstown district, Eastern Cape Province, South Africa (n=10), and slaughtered at The Grahamstown Ostrich Abattoir. The birds ranged in age from 22 months to five years. Samples were collected approximately 60 minutes after the birds had been slaughtered. Drops of semen were gently squeezed from the distal ductus deferens into test tubes containing 2.5% glutaraldehyde in 0.13M Millonig‟s phosphate-buffer. Smears for light microscopy (LM) were prepared from the fixed cell suspensions, air-dried and stained with Wrights‟ stain (Rapidiff®, Clinical Sciences Diagnostics, Johannesburg, South Africa). The dried smears were fixed in methanol for 20 seconds, stained with eosin for 30 seconds, blotted and then stained with methylene blue solution for 60 seconds. The smears were then gently rinsed with distilled water and allowed to dry before mounting with Entellan® and a coverslip. Smears from each bird were examined with an Olympus BX63 light microscope (Olympus Corporation, Tokyo, Japan) using a 100x oil immersion objective (bright field as well as phase contrast microscopy) to evaluate sperm morphology and determine the incidence of sperm defects by counting the number of normal/abnormal sperm present in a total of 300 cells. Images of sper cells were digitally recorded. The linear dimensions of the various segments of the sperm (acrosome, nucleus, midpiece, principal piece and end piece), as well as the total length of the cells, were determined using phase contrast light microscopy. A minimum of 20 cells from each of the ten Grahamstown birds and the five Rustenburg birds were measured. The measurements were processed using the Soft Imaging System iTEM software (Olympus, Műnster, Germany) and expressed as the average ± SD. Statistical analysis of the different parameters assessed was performed using a computer package (Microsoft Office Excel 2010) and descriptive statistics for each of the sperm segments as well as the total sperm length were generated. The two groups of birds (Grahamstown and Rustenburg) were further compared using the Student‟s two-tailed unpaired t-test with a 95% probability to determine if there were any statistical differences between the two groups.
Electron microscopy
Samples for transmission (TEM) and scanning electron microscopy (SEM) were fixed overnight at 4ºC in 2.5% glutaraldehyde in 0.13M Millonig‟s buffer, pH7.4. Using gentle centrifugation and resuspension throughout, the samples were washed in Millonig‟s phosphate buffer, pH 7.4, before post-fixation in similarly buffered 1% osmium tetroxide for one hour. After two subsequent washes in buffer, the samples were dehydrated through a graded ethanol series (50%, 70%, 90%, 96%, 3x 100% for 20 minutes each) and cleared with propylene oxide for 20 minutes before embedding in epoxy resin (TAAB 812 resin; TAAB Laboratories, England). Thin sections were cut with a Reichert-Jung Ultracut (C. Reichart AG., Vienna, Austria) ultramicrotome using a diamond knife and stained with lead citrate and uranyl acetate before being viewed in a Philips CM10 transmission electron microscope (Philips Electron Optical Division, Eindhoven, The Netherlands) operated at 80kV.
Chapter 1 General introduction and basic architecture of the emu male reproductive tract
Chapter 2 Morphology of normal sperm
Chapter 3 Spermiogenesis
Chapter 4 A novel transient structure associated with spermiogenesis
Chapter 5 Incidence and morphological classification of abnormal sperm
Chapter 6 Morphology and origin of abnormal sperm I: Head base bending and disjointed sperm
Chapter 7 Morphology and origin of abnormal sperm II: Abaxial tail implantation
Chapter 8 Morphology and origin of abnormal sperm III: Multiflagellate sperm
Chapter 9 Morphology and origin of abnormal sperm IV: Miscellaneous defects
Chapter 10 General conclusions
