Evaluation of buffalo semen by Trypan blue/Giemsa staining and related fertility in vitro L. Boccia, R. Di Palo, A. De Rosa, L. Attanasio, E. Mariotti, B. Gasparrini 1
DISCIZIA, Faculty of Veterinary Medicine, “Federico II” University, Via F. Delpino, 1, Naples, Italy
Corresponding author: L. Boccia. DISCIZIA,“Federico II” University, Naples, Italy - Tel. 0039 0812536494 - Fax: 0039081292981 - Email:
[email protected]
abstract: The aim of this work was to verify the feasibility of an easy, quick double staining technique for evaluation of frozen-thawed semen to predict the fertilizing capability in vitro of buffalo bulls. In Experiment 1, frozen-thawed semen from 6 bulls was stained with double Trypan blue/ Giemsa and the incidence of acrosome-intact live (AIL), acrosome-intact dead (AID), acrosome-lost live (ALL) and acrosome-lost dead (ALD) sperm was recorded. In Experiment 2, sperm from the same bulls were used to fertilize in vitro matured oocytes. The data obtained confirm that there is a strong “bull effect” in buffalo species, with differences in the percentage of AIL sperm at thawing, in cleavage and blastocyst rates among bulls. Interestingly, it was found that this staining technique can be used for a preliminary screening to select semen to use for IVF, as shown by the correlation existent between the percentages of acrosome-intact viable sperm cells at thawing and the blastocyst yields for 4/6 bulls. Key words: Semen, Buffalo, Trypan blue/giemsa, Quality evaluation. INTRODUCTION - The increased demand from different Countries of embryos derived from buffaloes of superior genetic make up currently faces a profound limitation in the difficult selection of truly superior bulls which can also offer a good in vitro fertility. A major factor affecting the diffusion in the field of both artificial insemination and the technology of in vitro embryo production in buffalo species, is the high variability of the fertilizing ability of buffalo bulls. Because only few bulls have good fertilizing capability in vitro (approximately 10 %, Gasparrini 2002), an accurate screening of sperm of several bulls is required in order to identify a semen suitable for IVF programs. It is known that a more accurate estimation of the fertility of an ejaculate is reached by increasing the number of tests performed in the laboratory. Nevertheless, it may occur that a semen judged suitable on the basis of fertility parameters tested in the lab, gives poor results after IVF. Therefore, currently the most accurate screening still goes through IVF trials, with different bulls tested on the same batch of eggs, that, in this species, because of the poor number of oocytes usable, are time-consuming. Therefore, the identification of a quick and reliable method for predicting the fertilizing capability of sperm from different bulls has great importance in this species. A double Trypan blue/Giemsa staining method was first used for sperm evaluation by Kovács and Foote in 1992. The application of this staining technique is very simple, rapid and allows to examine the results even after many years. This procedure can be used to assess sperm Ital.J.Anim.Sci.
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VIII World Buffalo Congress viability evaluating the membrane status of sperm cells subdomains (head, acrosome, tail) through different staining of the viable and not viable segments. It is possible to differentiate different classes of spermatozoa, on the basis of the characteristics of the head (live or dead), of the tail (intact, lost or damaged) and of acrosome. Sperm cells displaying an intact viable head and acrosome but trypan blue stained tail were evaluated as non-viable (Nagy et al., 1999). This technique was successfully employed to assess viability and acrosome status in water buffalo spermatozoa (Presicce et al., 2003). The aim of this study was to verify the feasibility of Trypan blue/ Giemsa staining to predict the in vitro fertilizing capability of different buffalo bulls. In particular, the objectives were: 1) in Experiment 1, to evaluate semen quality of 6 buffalo bulls by the double staining Trypan blue/Giemsa; 2) in Experiment 2, to assess the fertilizing capability in vitro of sperm from the same 6 bulls. MATERIAL AND METHODS - Experiment 1 - Fixation and staining of spermatozoa: Frozen semen from 6 Italian water buffalo bulls was thawed and a 10x dilution was prepared by adding 1 part of semen to 9 parts of 0.9% NaCl. One drop of 0.27 % trypan blue, made from two parts of a buffered 0.4% solution (Sigma T8154) and one part of 0.9% NaCl and one drop of diluted semen were mixed on a slide and over the entire surface by using another slide, both held parallel to each other. Two smears were thus prepared by using a single semen droplet. Slides were air-dried in a near vertical position then put into a fixative in a jar for two minutes, and then rinsed with tap and distilled water. The fixative consisted of a mixture of 86ml 1 N HCL and 14 ml of 37% formaldheyde solution with the addition of 0.2g Neutral Red (Sigma N-2880). Slides were put into jars containing the Giemsa staining solution and left overnight (16 to 20 hours) at room temperature. The Giemsa Staining solution was freshly prepared by adding 7.5% (v/v) of Giemsa stock solution (Sigma GS-500) to distilled water. Slides were rinsed again in tap and distilled water for 2 minutes, air-dried in a near vertical position and coverslipped with Entellan (Merck 107). Microscopic evaluation of sperm cells (N= 3298) were performed at 40x and 100x oil immersion magnification. Based on staining characteristics of sperm cells we differentiated four categories: acrosome-intact live (AIL), acrosome-intact dead (AID), acrosome-lost live (ALL) and acrosome-lost dead (ALD). We recorded as live only the sperm displaying both head and tail viable and as dead those with either the head or the tail unviable. Experiment 2 - Oocyte collection and in vitro maturation - Buffalo ovaries were collected from an abattoir and transported to the laboratory in physiological saline supplemented with 150 mg/L kanamycin at approximately 30-35°C, within 3–4 h after slaughter. Cumulus– oocyte complexes (COCs) were recovered by aspiration of 2–8 mm follicles using an 18-G needle under vacuum (40–50 mmHg). The COCs with a compact, non-atretic cumulus and a homogeneous cytoplasm were selected. The COCs selected were washed thoroughly in Hepes-buffered TCM199 with 10 % fetal calf serum (FCS) and allocated to 50 µl drops (10 per drop) of the in vitro maturation (IVM) medium, consisting of TCM199 buffered with 25 mM sodium bicarbonate and supplemented with 10% FCS, 0.2 mM sodium pyruvate, 0.5 µg /mL FSH, 5 µg /mL LH, 1 µg /mL 17β-estradiol, 50 mg/mL kanamycin and 50 µM cysteamine and 0.3mM cystine (Gasparrini et al 2006). IVM was carried out at 38.5 °C for 20-22 h in a controlled gas atmosphere consisting of 5% CO2 in humidified air. In vitro fertilization and culture - Spermatozoa were prepared from frozen–thawed semen, and treated by swim-up procedure in Hams medium for 1 h. The pellet obtained after centrifugation of the supernatant was re740
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VIII World Buffalo Congress suspended to a final concentration of 2 x 106 mL -1 in the fertilization medium, consisting of Tyrode albumin lactate pyruvate (Lu et al., 1987) supplemented with 0.2 mM penicillamine, 0.1 mM hypotaurine and 0.01 mM heparin. Insemination was performed in 50 µl drops of fertilization medium under mineral oil (5 oocytes per drop) at 38.5 °C under humidified 5% CO2 in air. Twenty hours after IVF putative zygotes were removed from the fertilization medium, stripped of cumulus cells by gentle pipetting, washed twice in a Hepes-buffered synthetic oviduct fluid (SOF) medium and allocated to 20 µl drops of SOF including essential and nonessential amino acids and 8 mg/mL bovine serum albumin (Tervit et al., 1972). Culture was carried out under humidified 5% CO2, 7% O2 and 88% N2 at 38.5 °C. Differences in the percentages of different sperm categories at thawing (Experiment 1) and in cleavage and blastocyst (BL) rates (Experiment 2) among bulls were analyzed by Chi Square test. RESULTS AND CONCLUSIONS - The results of the Experiment 1 are shown in Table 1. In particular, the percentage of AIL sperm at thawing, that is an index of sperm viability, was significantly different among bulls, with higher (P
