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BERAN JAN, ŠIMONÍK ONDŘEJ, STÁDNÍK LUDĚK, RAJMON RADKO, DUCHÁČEK JAROMÍR, KREJCÁRKOVÁ ADÉLA, DOLEŽALOVÁ MARTINA, ŠICHTAŘ JIŘÍ: Eff ect of bull, diluter and LDL-cholesterol concentration on spermatozoa resistance against cold shock. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 2013, LXI, No. 6, pp. 1575–1581 The objectives of this study were to determine and evaluate the eff ect of bull, diluter and addition of LDL in diff erent concentration on the percentage rate of spermatozoa survival a er cold shock. In total, four bulls were collected during a period of eight weeks. A total of 8 samples of fresh semen with required quality were processed. Three extenders were used for dilution of each sample; AndroMed®, Bioxcell® and Triladyl®, each in standard and LDL enriched variants. In the case of AndroMed® and Bioxcell®, 4, 6 and 8% of LDL were simply added. In Triladyl®, 6, 8 and 10% of LDL replaced the standard egg yolk component. Resistance of spermatozoa against cold shock (0 °C, 10 minutes) was evaluated by the percentage rate of live sperm using Eosin-Nigrosine staining immediately and 2 hours a er heat incubation (37 °C). The results showed the infl uence of bull individuality as an important factor. Among diluters used it is possible to recommend AndroMed® and Bioxcell® due to signifi cantly (P < 0.01) lower decline of live sperm proportion during the cold shock test than Triladyl® (−9.19, respectively −4.95%). The optimal LDL concentration increasing resistance of spermatozoa against cold shock was not determined, therefore subsequent research is necessary. bull semen, sperm survival, cold shock, extender, LDL cholesterol Fertility of dairy cows has declined over the past fi ve decades as milk production per cow has increased (Zink et al., 2012; Walsh et al., 2011). The issue of cattle fertility was intensively studied in recent years from cow point of view and eff ects on their reproductive performance (Doležalová et al., 2013; Roche et al., 2011; Hanuš et al., 2010; LeBlanc et al., 2010). However, cow’s reproduction results are also aff ecting by male component which is represented by the fertilization ability of bull ejaculate. Sperm quality is infl uenced by many factors, e.g. by such internal factors as breed, variation between individuals, and age of sire (Beran et al., 2011; Štolc et al., 2009), and by such external factors as environmental conditions (Balic et al., 2012), composition of the diet (Horký et al., 2012) and frequency of collecting ejaculate (Kaya et al., 2002). Collection of ejaculate and its subsequent processing present further potential risk factors for declined sperm quality. Phases of producing insemination doses (diluting of sperm, fi lling of straws, cooling and freezing) have a signifi cant eff ect on sperm motility a er thawing (Siddique et al., 2006), and the properties of extender used are especially important (Beran et al., 2012; Hegedüšová et al., 2012), as well as method of thawing (Filipčík and Hanuláková, 2011). Low Density Lipoprotein (LDL cholesterol – LDL) – a component of egg yolk – is believed to 1576 Jan Beran, Ondřej Šimoník, Luděk Stádník, Radko Rajmon, Jaromír Ducháček, Adéla Krejcárková, Martina Doležalová, Jiří Šichtař be largely responsible for the egg yolk protective eff ects on spermatozoa during the freezing process (Pace and Graham, 1974) increasing their resistance against cold shock (Moussa et al., 2002). There is an assumption that extenders containing LDL, which is extracted from egg yolk, can have better eff ects on spermatozoa membranes during the freezing than diluters commercially exploited (Moussa et al., 2002). Biological tests of ejaculate have been developed to assess the resistance and the fertilization ability of sperm using LDL addition into diluters, e.g. shortterm heat test of sperm survival (Maurya and Tuli, 2003), hypo-osmotic swelling tests (Padrik et al., 2012), however resistance of sperm against cold shock has not been evaluated as well in relation to addition of LDL. Thus, the aim of this paper was to determine the eff ect of bull, diluter and addition of LDL in diff erent concentration on the percentage rate of spermatozoa survival a er cold shock. MATERIAL AND METHODS Semen collecting, dilution and processing Ejaculate of four bulls (No. 1, 2, 3, and 4) belonging to one artifi cial insemination (AI) centre approved for public use, were sampled and evaluated. Samples of ejaculate were obtained using an artifi cial vagina during the period of eight weeks and tested immediately a er collection. Volume of semen (VOL), density of sperm (DEN, and percentage of progressive motile spermatozoa above head (ACT) were evaluated by only one trained technician of AI centre. A total of 8 samples of fresh semen with required quality (minimum progressive motility 70% and sperm concentration 0.7 × 106 mm−3) were then transferred at 4 °C to the university laboratory within one hour for next processing. Three extenders were used for dilution of each sample; AndroMed®, Triladyl® (both from MiniTüb GmbH, Tiefenbach, Germany) and Bioxcell® (IMV, L’ Aigle, France). Control variants of diluters (0% LDL addition) were prepared according to the manufacturer’s instructions. Experimental variants included 4, 6 and 8% LDL addition into the AndroMed® and Bioxcell® diluters, and 6, 8 and 10% LDL addition into the Triladyl® diluter prepared without using egg yolk, which is normally its essential component. Higher concentrations of LDL in Triladyl® replaced cryoprotective properties of egg yolk. The extenders were prepared on the day of sampling and stored at the cooling box (4 °C) before ejaculate dilution. Samples of semen were pipetted using a sterile pipette to a sterile chilled (4 °C) tubes. Each sample of semen was immediately diluted to 50 000 spermatozoa/ml. The required amount of diluters was applied using sterile syringes directly to the tubes with samples. Therea er tubes were sealed with sterile stoppers, mixed gently and placed into the cooling box (4 °C). Evaluation of resistance against cold shock Three capillaries (0.1 ml) were successively fi lled from each sample at 4 °C, closed at one end with plasticine and stored at 0 °C into a cooled bath (No Ice, Bibby Scientifi c, Ltd., Staff ordshire, UK) for ten minutes. A er the end of cold incubation the capillaries content was gently mixed on preheated hour glass (37 °C) with 20 μl of Eosin by circular motion for 30 sec. and then Nigrosine was added at amount of 40 μl. A volume of 20 μl of the resulting suspension was added into a preheated glass slide and smear was done at the beginning of the test (time 0). This procedure was repeated a er 2 hours heat incubation of extended semen samples in a water bath at 37 °C. A er drying each smear (72 of each collecting day, 576 together) was examined under a phase contrast microscope (Eclipse E200, Nikon®, Tokyo, Japan) at 1 000x magnifi cation and with oil immersion by only one evaluator. Minimum of 100 spermatozoa was classifi ed as either dead (with red heads) or live (with white heads) and expressed as a percentage rate of live sperm at the beginning of the test (L0) and a er 2 hours of heat incubation (L2). Statistical analysis The data were evaluated with statistical so ware SAS 9.3. (SAS/STAT® 9.3, 2011) using UNIVARIATE, CORR, and MIXED procedures. The following equation was used: Yijkl = μ + BULLi + DILj + LDLCk + b1*(VOL) + + b2*(DEN) + b3*(ACT) + eijkl, where: Yijkl observed value of the dependent variable (percentage rate of live sperm at the beginning of the test and a er 2 hours heat incubation, diff erence between these two measurements), BULLi fi xed eff ect of the i th bull (i = 1, n = 216; 2, n = 216; 3, n = 72; 4, n = 72); DILj fi xed eff ect of the j th diluter (j = 1 – AndroMed®, n = 192; 2 – Bioxcell®, n = 192; 3 – Triladyl®, n = 192); LDLCk  fi xed eff ect of the k th concentration of LDL (k = 0, n = 144; 4, n = 96; 6, n = 144; 8, n = 144; 10, n = 48); b1*(VOL) ....regression on volume of ejaculate; b2*(DEN) ....regression on density of sperm; b3*(ACT) ....regression on activity of sperm; eijkl residual eff ects. The diff erences between the variables estimated were tested at the levels of signifi cance P < 0.05 and P < 0.01. Pearson correlation coeffi cients were also determined. RESULTS AND DISCUSSION The basic statistical characteristics of observed data are shown in Tab. I. The volume of ejaculate Eff ect of bull, diluter and LDL-cholesterol concentration on spermatozoa resistance against cold shock 1577 ranged from 6.2 to 11.8 g in selected bulls. The volumes of ejaculates correspond to the fi ndings of other authors, e.g. Louda et al. (2007) reported the range 3–12 g in sires kept in AI centre. Ball and Peters (2004) mentioned the closer range from 5 to 6 g. The sperm density ranged from 0.9 to 1.8 × 106 mm−3. Our results again agreed with those of Louda et al. (2007), who reported standard sperm density of bulls’ ejaculates from 0.8 to 2.0 × 106 mm−3. Ball and Peters (2004) observed a range from 0 in azoospermic bulls to 3 000 × 106 mm−3 in excellent sires, however especially mentioned substandard marginal bulls could not be used as widely applied sires. The activity of sperm immediately a er collection ranged from 80 to 90% due to determined threshold of 70% commonly used in Czech AI centers. These values correspond with those of Louda et al. (2007), who reported sperm activity 45–75% or more. Similarly, Ball and Peters (2004) reported that at least 60% of the spermatozoa should be shown straight progressive movement above head. This requirement was fulfi lled within our observation. Cold shock test belongs to the main evaluation method of sperm quality. We take a refl ection about viability and fertility of ejaculate. The rate of live sperm ranged from 23.27 to 93.2% in the beginning and from 5.98 to 89.25% a er 2 hours of the heat i

Effect of bull, diluter and LDL-cholesterol concentration on spermatozoa resistance against cold shock