Megha Pande

Enriching membrane cholesterol improves stability and cryosurvival of buffalo spermatozoa

Buffalo spermatozoa are comparatively more susceptible to freezing hazards than cattle spermatozoa. In recent times incubation of spermatozoa with cholesterol-loaded-cyclodextrins (CLC) has shown improvements in semen quality in several species. Therefore, this study was undertaken to evaluate the incubation level of CLC at which maximum benefit is derived for the buffalo spermatozoa. For the study, 120 million spermatozoa were incubated in 2, 3 and 4 mg/mL of CLC (Gr II, III and IV, respectively) and cholesterol and phospholipids content, their ratio, flow cytometric evaluation of plasma membrane integrity (PMI), plasma membrane fluidity and extent of cryoinjury (Chlortetracycline, CTC assay) were compared with an untreated control (Gr I). Additionally the ability of cholesterol-loaded-spermatozoa to undergo induced acrosome reaction (IAR) using ionophore calcium (A23187) was evaluated in frozen-thaw samples. Data show a significant and linear increase (CV=0.88) in cholesterol content of spermatozoa in Gr II, III and IV and a significant decrease in phospholipids content at frozen-thaw stage in Gr IV than Gr III spermatozoa. The study revealed a significant improvement in PMI and significant reduction in plasma membrane fluidity and cryoinjury of CLC treated spermatozoa at progressive stages in three groups compared to control. Nevertheless, spermatozoa of Gr II, III and IV were significantly less responsive to ionophore calcium (A23187) than Gr I. This study shows for the first time that incubation of buffalo bull spermatozoa with CLC (3mg/120×10(6)) prior to processing permits greater numbers of sperm to survive cryopreservation while allowing spermatozoa to capacitate and the acrosome to react to AR inducer ionophore calcium (A23187).

Determination of Mitochondrial Function in Sperm Cells

With the advancement of science, detecting functionality of mitochondria has become one of the desirable parameters to evaluate sperm quality. This is further aided by availability of wide spectral range of fluorescent probes that has advantage of simultaneous multi-parametric assays. Apart from application of fluorescent probes, computer-assisted image analyzers can be used to assess mitochondrial functionality via motility attributes. In this chapter, we have attempted to describe evaluation of mitochondrial functional status using fluorescent dyes and thus have listed relative merits, protocols, and what to look for in the stained sperm sample. This important chapter also includes triple staining of sperm cells to elucidate integrity of acrosome, plasma membrane, and mitochondrial functions simultaneously. Additionally, procedure for flow cytometry of fluorescent-stained sperm cells as an objective method and tetrazolium (MTT) reduction assay as an easy, inexpensive, and rapid spectrophotometric protocol to determine mitochondrial function in spermatozoa is described.

Immuno-Reproduction Assays in Semen Biology

The practical advantage of an immunological methodology to the study of carbohydrates, proteins, and other cellular structures is that when carefully carried out, results are highly specific, sensitive, and repeatable. All immunological assays in semen biology involve measurement of the interaction of targeted antibody/antigen (Ab-Ag) with that of Ag-Ab. Since most of the investigations in the semen biology involve either quantitative or qualitative assay of interaction of Ab with Ag, we have divided such assays in two parts. Part one, the qualitative assays, includes the enzyme-linked immunosorbent assay (ELISA) with its four common variants, namely, indirect ELISA, direct competitive ELISA, antibody-sandwich ELISA, and radioimmunoassay (RIA). Part two, the quantitative assays, includes double immunodiffusion (DID), Western blot, and dot blot assays. Additionally, tests for screening of antisperm antibody (ASA) on the sperm cells as well as biological fluids have been explained. This chapter also includes isolation of Ab (specifically IgG) by chromatography method. The comparison of various assays has been provided in the relevant sections.

Estimating Metabolic Activity of Spermatozoa

The motility of live spermatozoa indicating energy requirement and thus presence of intrinsic metabolic pathways in the cell has led researchers to speculate relationship of sperm metabolism with semen quality parameters. Over the period, many assays, some quantitative and some qualitative, to estimate metabolic aspects of sperm life have been developed. Investigators have reported significant relationship of metabolic assays with that of sperm concentrations and motility. Moreover, a significant relationship of resazurin reduction assay with that of oxidative stress of spermatozoa has also been reported recently. This chapter outlines principle and procedures involved in various assays employed for estimating metabolic rates of bovine spermatozoa. Modification of resazurin assay to objectively measure colour changes using spectrophotometer and, in fructose estimation, protocols to evaluate metabolic rates in sperm suspension and frozen-thawed samples have been included.

Selection of Spermatozoa

The isolation of mammalian spermatozoa from the surrounding seminal fluid is a crucial practice commonly applied in assisted reproductive technology applications. The selection of sperm isolation protocol is critically important for investigators, for clinicians preparing sperm samples to be used in reproductive biotechnologies, in veterinary andrology laboratories, and in animal husbandry. Considering the growing importance of the sperm selection techniques, this chapter deals with established sperm selection techniques, viz., simple washing of spermatozoa, swim-up, and discontinuous density-gradient protocol. In addition as a corollary to these protocols, techniques to recover spermatozoa from the epididymis or testicular tissues have been provided alongside.

Evaluating Resistance of Spermatozoa to Adverse Conditions

The detrimental effects of cryopreservation on sperm function and fertility have been widely studied in bovine. Accumulated data show great variation in response of spermatozoa to stress of temperature variation in ejaculates from same bull (within bull) as well as between bulls. Moreover, many a times ejaculates are not processed for long-term storage but are used as liquid semen for varied reasons. To maintain optimum level of quality, such semen samples need to be tested regularly, in a rapid and cost-effective manner for their ability to withstand adverse storage conditions and suspension medium as compared to neat semen. This chapter outlines assays such as cold shock resistance test to measure robustness of fresh spermatozoa under fluctuating temperature conditions, incubation assays to evaluate ability to withstand changes in tonicity and post-thaw motility-ageing assay to approve quality of cryopreserved semen. A comparison of the advantages of assays outlined above has also been provided.

Determination of Capacitation-Related Changes Using Fluorescent Stains

The capacitation followed by acrosome reaction is a prerequisite for spermatozoa to acquire fertilizing ability. This is evidenced by a strong relationship between fertility, post-thaw motility, and percent non-capacitated spermatozoa. Therefore, it is important to analyze sample simultaneously for sperm viability and normalcy of the acrosomes. Though capacitation status of the spermatozoa can be effectively evaluated in fixed samples using differential interference contrast microscopy, a classical approach, this technique suffers from subjective variation and time factor. On the other hand, evaluation of capacitation status can also be carried out using various fluorescent probes, viz., chlortetracycline, merocyanine-540, LysoTracker, anti-acrosin antibodies, and propidium iodide, to name a few. Fluorescent probes have added advantage of flow cytometer evaluation, which is rapid and more accurate than classical methods. Also, combination of protocol for chlortetracycline probe with that of a viability stain, viz., ethidium homodimer, has been included for simultaneous evaluation of capacitation status and viability of spermatozoa. This chapter evaluates protocols of fluorescent staining of spermatozoa, including triple staining, and compares merits and demerits of each probe.

Estimates of Sperm Motility

Spermatozoa motility is a very important attribute most commonly exploited for discriminating between a good and a bad semen sample at the fresh as well as post-cryopreservation stage. Accurate measurement of motility provides crucial information about viability of spermatozoa as well. In the chapter on sperm motility, principles governing various motility parameters, protocols and finer practical hints have been provided for the investigator to carry out experimentation accurately. We have provided protocols of estimating sperm motility using CASA for an effective and objective assessment as well. This section includes various advantages and disadvantages of CASA vis-a-vis other motility parameters as well.

Counting Sperm Numbers

Selection of the best ejaculate from the donor species depends greatly on precise calculation of the concentration of the live sperm cells. Different laboratories engaged in the semen biology work employ variety of instruments and procedures. These, coupled with the variability of the result induced by the objective assessment (inter-technician variation), make it difficult for an investigator to decide on the suitability of an assay for his work. This chapter addresses this dilemma by listing protocols of various assays for determination of concentration of spermatozoa and their relative merits. Thus, procedures of haemocytometer, spectrophotometer and NucleoCounter SP-100 are covered. Since the flow cytometer and CASA have been described elsewhere, their procedural details are not explained here.

Evaluating Sperm Cell Viability and Membrane Integrity

Rapid and precise assessment of sperm viability is vital in evaluating seminal quality. The viability (%) is determined by identifying sperm with an intact cell membrane, either by carrying out dye exclusion tests or by hypo-osmotic swelling. In this chapter, we have attempted to cover some important assays with the underlying principles including assay based on the hypotonic swelling and its variations as an indicator of the biochemical integrity of the membrane. This chapter contains procedures related to hypo-osmotic swelling and application of supravital dye and various fluorescent dyes such as Hoechst 33342, DAPI, PI and SYBR-14 including their combination for determining spermatozoa viability. A comparison of the assay vis-a-vis their merit has been provided.