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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Sperm fertility and viability following 48 h of refrigeration: Evaluation of different extenders for the preservation of bull semen in liquid state A.M. Crespilho a,∗ , M. Nichi b , P.N. Guasti c , C.P. Freitas-Dell’Aqua c , M.F. Sá Filho b , R.R. Maziero c , J.A. Dell’Aqua Jr c , F.O. Papa c a b c

School of Veterinary Medicine, Santo Amaro University, UNISA, São Paulo, Brazil Department of Animal Reproduction, São Paulo University, USP, São Paulo, Brazil Department of Animal Reproduction and Veterinary Radiology, São Paulo State University, UNESP, Botucatu, Brazil

a r t i c l e

i n f o

Article history: Received 8 August 2013 Received in revised form 27 February 2014 Accepted 28 February 2014 Available online xxx

Keywords: Bull Semen extender Soy lecithin Sperm refrigeration Lipid peroxidation

a b s t r a c t Two experiments were conducted to compare the effectiveness of different extenders conventionally used for semen cryopreservation to maintain the viability and fertility of cooled bull semen. In Experiment 1, sperm samples obtained from 20 Nellore bulls were preserved at 5 ◦ C for 48 h using two extenders containing 20% of egg yolk [Tris (TRIS-R) and Botu-Bov® (BB)] and another composed of 1% soy lecithin [Botu-Bov® -Lecithin (BB-L)] as substitutes for animal origin products. The samples were evaluated at 6, 24 and 48 h for plasma and acrosomal membrane integrity, quantification of thiobarbituric acid reactive substances (ng of TBARS/108 cells) and sperm motility parameters by computer-assisted semen analysis (CASA). In Experiment 2, pregnancy rate (P/AI) of 973 fixed-time artificially inseminated Nellore cows were compared when cows were inseminated with conventionally cryopreserved semen in TRIS-egg yolk glycerol (TRIS-C Control, n = 253) or semen cooled for 48 h in TRIS-R (n = 233), BB (n = 247) or BB-L (n = 240). Although none of the extenders used was effective on maintaining total progressive motility and cellular integrity throughout the 48h of the refrigeration period (P < 0.01), BB-L conferred greater protection against oxidative stress (P < 0.05) than egg yolk-based medias. The P/AI for semen samples preserved in TRISC, TRIS-R, BB and BB-L were 39.92a , 25.32b , 26.32b and 33.33ab , respectively. These results demonstrate that the three conventional extenders used for semen cryopreservation do not provide the protection required to maintain bull semen fertility under refrigeration for a 48h period, resulting in reduced pregnancy rates. However, the use of lecithin-based medium instead of egg yolk results in greater protection against lipid peroxidation, producing P/AI results comparable to those obtained using frozen semen. © 2014 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author at: Rua Professor Enéas de Siqueira Neto, 340, Jardim das Embuias, Postal Code: 04829-300, São Paulo, Brazil. Tel.: +55 11 2141 8500; fax: +55 11 2141 8500. E-mail addresses: [email protected], [email protected] (A.M. Crespilho).

Despite the importance and widespread use of frozen semen in cattle production, few advances in the development of new extenders have emerged in the past decades. A significant decrease in the integrity and functionality of spermatozoa is observed when semen is subjected to

http://dx.doi.org/10.1016/j.anireprosci.2014.02.020 0378-4320/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Crespilho, A.M., et al., Sperm fertility and viability following 48 h of refrigeration: Evaluation of different extenders for the preservation of bull semen in liquid state. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.020

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conventional cryopreservation protocols (Celeghini et al., 2008). Freezing is responsible for a significant decrease in sperm motility (Chaveiro et al., 2006; Thomas et al., 1998), leading to several biochemical and structural alterations that may affect different cytological compartments of the spermatozoa, causing most of the cells to die during processing (Yoshida, 2000). The decrease in spermatozoa viability and fertility associated with cryopreservation has motivated several studies to address preserving semen in a liquid state for several species including cattle (Bucher et al., 2009; Crespilho et al., 2012a) antelope (Adeel et al., 2009), horses (Crespilho et al., 2013), goats (Purdy et al., 2010) and sheep (O’Hara et al., 2010), demonstrating promising results for sperm viability preservation. The main advantage of the cooled semen process is that it prevents damage associated with freezing, thereby ensuring greater sperm viability. This allows the insemination dose to be reduced, thereby optimizing the use of sires of high genetic merit in artificial insemination programs (Bucher et al., 2009; Verberckmoes et al., 2005). The success of semen preservation in the liquid state requires that the reduction in sperm motility and metabolic activity caused by refrigeration be reversible (Yoshida, 2000). Considering that storage time influences the viability of cooled semen (Batellier et al., 2001), one of the main uses of cooled bull semen is in fixed-time artificial insemination (FTAI) programs. These protocols allow the insemination of a large number of animals in a short time interval (Bucher et al., 2009), making it possible to incorporate semen refrigeration technology despite the short longevity of these samples. Thus, cooled bull semen represents a promising alternative to cryopreserved semen that may increase pregnancy rates (P/AI) and decrease the overall cost of FTAI programs. In a recent case report, the conception rate obtained after AI using the cooled semen for 24 h was 15% greater than conventional frozen-thawed bull semen, demonstrating the usefulness of cooled semen as a strategy to enhance the fertility of FTAI programs in beef cattle (Crespilho et al., 2012a). However, because most of the studies involving cooled bull semen were conducted prior to the arrival of cattle estrous cycle synchronization protocols (Almquist and Wickersham, 1962; Blackshaw et al., 1957; Foote, 1962, 1970; O’Dell et al., 1959), the impact of cooled bull semen on the pregnancy rates in fixed time artificial insemination programs have not been addressed in the literature. One of the main factors associated with the decrease in the motility and fertility of refrigerated sperm is the production of reactive oxygen species (ROS), which occurs as a normal consequence of sperm metabolism and results in an irreversible decrease in the quality of cooled semen (C¸oian et al., 2010). Considering that spermatozoa have a limited capacity to resist oxidative stress (Nichi et al., 2006), it is essential that extenders minimize the deleterious effects of ROS. The objective of the present study was to test the effectiveness of various extenders usually used for the cryopreservation of sperm at maintaining the viability and fertility of bull semen stored under refrigeration

temperature. This study also tested the hypothesis that the use of semen subjected to refrigeration for a 48-h period may increase the P/AI ratios in fixed-time artificial insemination programs in beef cows synchronized with a progesterone and estrogen-based protocol. 2. Materials and methods 2.1. Semen collection The methodology implemented in this study was approved (process number 228/2011) by the Ethics Committee in Animal Experimentation of the São Paulo State University, Brazil. Semen samples were obtained through electroejaculation from 20 Nellore bulls (Bos taurus indicus) aged 24–30 months. In Experiment 1, two ejaculates were obtained from each sire (the first intended for initial seminal quality analysis and a second sample for the experimental procedure). Minimum quality criteria for fresh semen to be included in the experimental procedure included sperm motility greater than 70%, percentage of major defects below 20%, percentage of minor defects below 20% and percentage of total defects below 30%. Five sires were selected for Experiment 2 based on the phenotypic characteristics of the breed and seminal quality criteria used in Experiment 1, and each animal was subjected to serial seminal samplings through electroejaculation (with a 2-day interval between samplings) for 15 days to ensure a period of biological leveling and adaptation to the new routine before beginning sample collection for the artificial insemination program. To produce the insemination doses a total of 10 ejaculates (2 ejaculates from each bull) were collected, according to minimum quality criteria adopted. 2.2. Semen processing Immediately after sample collection, the total motility and sperm vigor of the ejaculate samples were subjectively evaluated using light microscopy, and total sperm concentration was determined using a Neubauer hemocytometer chamber. 2.2.1. Experiment 1 Three extenders were used for semen refrigeration: Tris-egg yolk-fructose (TRIS; 30 g [Tris (hydroxymethyl) aminomethane], 17 g citric acid, 12.5 g fructose, 0.20 g amikacin sulfate, 2 mL Orvum Est Pastum and 20% egg yolk); Botu-Bov® (BB; Botufarma Ltda., Botucatu, São Paulo, Brazil) also containing 20% egg yolk in 1 L of final solution; and Botu-Bov-Soy Lecithin (BB-L; Botufarma Ltda., Botucatu, São Paulo, Brazil), in which the egg yolk was completely replaced by 1% lecithin, according to Crespilho et al. (2012b). All media were made in single fractions free of glycerol, and clarified by high centrifugation force (5000 × g for 1.5 h in refrigerated centrifuge). Each semen sample was fractionated into three equal aliquots immediately after sample collection, deposited into 50 mL plastic tubes, diluted in TRIS, BB or BB-L medium and packaged in 0.5 mL French straws (IMV® Technologies, L’Aigle Cedex, France) at a final concentration of

Please cite this article in press as: Crespilho, A.M., et al., Sperm fertility and viability following 48 h of refrigeration: Evaluation of different extenders for the preservation of bull semen in liquid state. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.020

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30 × 106 total spermatozoa/straw. The samples were stored in Botutainer® transport boxes (Botufarma Ltda., Botucatu, São Paulo, Brazil), which were used for passive refrigeration of the bovine spermatozoa at a constant temperature of 5 ◦ C for 48 h, using a previously tested cooled curve for bull semen (0.13 ◦ C/min until the stabilization temperature at 5 ◦ C). The effect of each medium used for refrigeration was evaluated in duplicate by computerassisted semen analysis (CASA, Hamilton Thorn Research IVOS-12, Beverly, USA), plasma and acrosomal membrane integrity measurements (PAMI, %) and the quantification of thiobarbituric acid reactive substances (ng TBARS × 108 ). 2.2.1.1. CASA (computer-assisted semen analysis). Samples were analyzed at 6, 24 and 48 h following the refrigeration process. Two straws per group were used for each evaluation. The straws were deposited in 1.5 mL plastic microcentrifuge tubes and pre-warmed in a dry bath at 37 ◦ C for 10 min before the analysis. CASA was used to evaluate 10 ␮L aliquots of semen in a Makler chamber prewarmed to 37 ◦ C. At least five random fields containing a minimum of 150 sperm/field were evaluated. The CASA technique measured total sperm motility (MOT, %), progressive motility (PM, %), curvilinear velocity (VCL, ␮m/s), amplitude of lateral head displacement (ALH, ␮m), linearity (LIN, %) and rapid motility (RAP, %). 2.2.1.2. Evaluation of plasma and acrosomal membrane integrity. Propidium iodide (PI) fluorescent primers and fluorescein-labeled Pisum sativum agglutinin (FITC-PSA) were used to analyze sperm viability using adaptations of the original protocols proposed by Way et al. (1995) and Celeghini et al. (2008). A 50 ␮L aliquot from each sample was transferred to a pre-warmed (37 ◦ C) 1.5 mL micro-centrifuge tube containing 50 ␮L of a 10 Mm/L sodium citrate solution. A 3 ␮L aliquot of PI (50 mg/mL in PBS solution) and 30 ␮L of FITCPSA (100 ␮g/mL in PBS solution) were added to the diluted semen. The samples were incubated at 37 ◦ C for 10 min prior to evaluation by epifluorescence microscopy (LEICA® , Solms, Germany) using a 540–525 nm fluorescence excitation filter and a 605–655 nm fluorescence emission filter. Two hundred spermatozoa (magnified 1000×) were evaluated for each sample, allowing the identification of four sperm subpopulations according to the fluorescence emission pattern: IPAM: intact plasma and acrosomal membranes; IPDAM: intact plasma and damaged acrosomal membranes; DPIAM: damaged plasma and intact acrosomal membranes; and DPDAM: damaged plasma and damaged acrosomal membranes. Only the proportion of IPAM (%) was considered for statistical analyses. 2.2.1.3. Quantification of thiobarbituric acid reactive substances (TBARS). Refrigerated semen samples were centrifuged at 2500 × g for 10 min, and 600 ␮L of the supernatant solution was deposited into a 2.0 mL microcentrifuge tube. Then, 1200 ␮L (1:2) of 10% trichloroacetic acid (TCA; 100 g in 1000 mL of water), prepared previously and stored at 5 ◦ C was added to each sample to promote protein precipitation in the supernatant solution according

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to Nichi et al. (2007). All samples were frozen in conventional freezers and stored at −18 ◦ C. Once thawed at room temperature, the samples were centrifuged again at 2500 × g for 10 min. The supernatant solution was collected, and 1 mL of the solution was transferred to a 5 mL glass tube to which 1 mL of 1% thiobarbituric acid (diluted in 0.05 M sodium hydroxide) was added; this solution was placed in a water bath at 100 ◦ C for 20 min. After boiling, the samples were cooled at 0 ◦ C in a container filled with ice. Thiobarbituric acid reactive substances (TBARS) were quantified using a spectrophotometer (UV–vis Spectrophotometer Ultrospec 3300 Pro, Biochrom Ltd., Cambridge, UK) calibrated to the wavelength of 532 nm. The results were compared to a previously established curve prepared with a standard malondialdehyde solution, which represents one of the main products of oxidative stress. The lipid peroxidation rate was expressed as ng of TBARS/108 sperm. 2.2.2. Experiment 2 Each sample was fractionated into four aliquots, which were placed into 50 mL plastic tubes and diluted in TRIS, BB or BB-L medium (following the same methodology described for Experiment 1) or in a Tris-egg yolk-fructose medium used for semen cryopreservation (TRIS-C, control group) at a final concentration of 6.4% glycerol according to Crespilho et al. (2012b). After packaging, the samples were refrigerated following the same previously described methodology. Semen parcels from the TRIS-C group were cooled using a digital refrigerator (Minitube® , Tiefenbach, Germany) at a constant temperature of 5 ◦ C for 4 h, then placed 5 cm from the liquid nitrogen (N2 ) surface inside a 40 L polystyrene box for 20 min and subsequently frozen by direct immersion in N2 and kept in a cryobiological container. The viability of the frozen and cooled bull semen was estimated on the day of its use in the FTAI program by subjective evaluation of total sperm motility (>50%) and the percentage of spermatozoa with morphological defects ( 0.20); thus, the method used for semen preservation was the only effect considered. Adjusted odds ratio (OR) and confidence interval (95%) values were generated through logistic regression analysis. The results are presented as probability of gestation in FTAI, assuming differences to be significant when P < 0.05 and trends when 0.05 < P ≤ 0.1. 3. Results 3.1. Experiment 1 Given that all semen samples (n = 20) were approved based on initial semen quality triage, Experiment 1 included ejaculates obtained from 20 different Nellore bulls with the results data for this experiment being included in Table 1. The total motility and percentage of rapidly moving sperm did not differ between the different treatments after 6 h of storage, and no interaction was observed between the initial refrigeration curve of the Botutainer® system and

Fig. 1. Effect of semen storage time at 5 ◦ C on lipid peroxidation, evaluated through the quantification of thiobarbituric acid reactive substances (ng of TBARS/108 cells). TRIS = Tris-egg yolk-fructose; BB = Botu-Bov® ; BBL = Botu-Bov® -Soy Lecithin

the evaluated extenders (P = 0.1921 and P = 0.3001, respectively). However, a decrease in MOT over the course of processing was observed (0.01 < P < 0.05) regardless of the extender in use (Table 1) for samples evaluated at 6, 24 and 48 h. A decrease in MOT over the initial 24 of the refrigeration process was only observed for samples diluted in TRIS (P < 0.05). Variables related to progressive movement (PM and LIN) differed among the different extenders at all the evaluated time points (Table 1). These variables also varied when considering each medium throughout the whole refrigeration period; greater decreases were observed for semen samples refrigerated in TRIS (PM, P < 0.0001; LIN, P = 0.0001). Pattern of plasma and acrosomal membrane integrity decreased throughout the refrigeration period (P < 0.0001), with a more pronounced decrease occurring over the initial 24 h of processing in all groups (P < 0.0001) and stabilization between 24 and 48 h relative to TRIS-diluted samples. Lesser rates of PAMI occurred with semen samples preserved in BB-L at all experimental time points when compared to BB and at 24 and 48 h when compared to TRIS. Lipid peroxidation was less for samples preserved in BB-L compared to those preserved in BB at 6 h (576.57 ± 76.42a compared with 1014.16 ± 185.98b , respectively; P = 0.0272) and was also less compared to those preserved in BB and TRIS at 24 h (BB-L = 447.60 ± 38.08a ; BB = 1470.69 ± 297.29b ; TRIS = 1589.56 ± 473.77b ; P = 0.0023) and 48 h (BB-L = 408.11 ± 29.58a ; BB = 1515.29 ± 250.05b ; TRIS = 1794.80 ± 313.27b ; P = 0.0011). The BB-L extender was the most effective at preventing oxidative stress throughout the 48-h refrigeration period, with no significant differences in TBARS values between 6, 24 and 48 h when using the soy lecithin-based medium (Fig. 1). However, TBARS values increased throughout the refrigeration period when BB (P = 0.0403) or TRIS (P = 0.0171), both of which contain 20% egg yolk, were used to maintain bull semen in the liquid state. 3.2. Experiment 2 There were sire effects (n = 5 bulls, P = 0.0007), batch of animals (P < 0.0001) and extender/methodology used for semen preservation (P = 0.0002) on C/AI when the

Please cite this article in press as: Crespilho, A.M., et al., Sperm fertility and viability following 48 h of refrigeration: Evaluation of different extenders for the preservation of bull semen in liquid state. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.020

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Table 1 Effects of different extenders (mean ± S.E.) on sperm motility and plasma and acrosomal membrane integrity (PAMI, %) throughout the 48-h storage period at 5 ◦ C. Sperm variable TRIS

MOT (%) PM (%) VCL (␮m/s) ALH (␮m) LIN (%) RAP (%) PAMI (%)

BB

BB-L

6

24

48

6

24

48

6

24

48

82.9 ± 2.1aA 52.8 ± 2.5aA 152.8 ± 4.2aA 6.3 ± 0.3aA 46.5 ± 1.3aA 79.6 ± 1.2aA 18.7 ± 2.2abA

74.7 ± 2.3aB 43.3 ± 2.2aB 170.7 ± 7.1aA 7.1 ± 0.3aA 42.5 ± 3.0aB 71.4 ± 0.7aB 4.0 ± 2.4aB

69.5 ± 3.1aB 38.1 ± 2.2aB 167.8 ± 6.4aA 6.9 ± 0.2aA 41.5 ± 0.6aB 65.6 ± 3.3aB 3.4 ± 3.7aB

82.9 ± 1.9aA 66.0 ± 1.9bA 116.3 ± 7.8bA 4.3 ± 0.3bA 61.9 ± 1.7bA 79.7 ± 1.9aA 20.7 ± 8.0aA

75.4 ± 2.1aAB 58.0 ± 2.4bB 125.1 ± 7.4bA 4.8 ± 0.3bA 57.6 ± 1.5bAB 71.6 ± 2.3aAB 4.2 ± 2.3aB

73.7 ± 2.8aB 56.6 ± 2.7bB 135.7 ± 6.2bA 5.2 ± 0.3aA 56.3 ± 1.6bB 69.2 ± 2.9aB 4.2 ± 3.8aB

73.8 ± 6.4aA 56.4 ± 5.1abA 149.5 ± 8.8abA 5.3 ± 0.3bA 55.8 ± 1.4cA 71.8 ± 6.5aA 12.8 ± 8.8bA

50.7 ± 7.0bAB 37.7 ± 5.5abAB 153.2 ± 14.7abA 5.8 ± 0.5bA 49.6 ± 3.1abA 48.5 ± 7.0bAB 2.3 ± 1.6bB

46.2 ± 7.5bB 33.8 ± 5.9aB 139.3 ± 13.1abA 10.4 ± 3.2aA 49.6 ± 3.4bA 45.3 ± 7.3bB 2.2 ± 2.8bB

a,b Lowercase letters on the same line indicate differences between treatments for each evaluated time point; A,B Uppercase letters on the same line compare the performance of each extender throughout the 48-h refrigeration period; MOT = total sperm motility; PM = progressive sperm motility; VCL = curvilinear velocity; ALH = amplitude of head displacement; LIN = Linearity; RAP = rapid spermatozoa; PAMI = plasma and acrosomal membrane integrity. TRIS = Tris-egg yolk-fructose; BB = Botu-Bov® ; BB-L = Botu-Bov® -Soy Lecithin.

initially proposed statistical model was used. However, no interactions between extenders and bulls (P = 0.7967) or extenders and batch of cows (P = 0.1143) were noted. Evaluation of the isolated effects of the extenders revealed the C/AI ratios of 39.92a , 25.32b , 26.32b and 33.33ab for semen samples preserved in TRIS-C (cryopreserved), TRIS-R, BB and BB-L (Table 2), respectively, resulting a greater probability of gestation when BB-L rather than the other extenders is used to preserve bull semen in liquid state under refrigeration. 4. Discussion All variables related to bull semen motility and progressive movement decreased significantly over the 48-h of refrigeration period regardless of the extender in use, consistent with the results obtained by Verberckmoes et al. (2005). Even though the decrease in temperature causes a significant decrease in sperm metabolism, reducing the rates of fructolysis and oxygen consumption (Blackshaw et al., 1957), sperm quality decreases throughout the refrigeration period regardless of the extender, dilution rate or storage conditions (O’Hara et al., 2010). Nevertheless, temperature exerts a significant influence on sperm survival throughout the refrigeration period (Batellier et al., 2001). Maintaining sperm in sub-optimal temperature conditions reduces sperm quality, sperm motility and plasma and acrosomal membrane integrity, and it also increases membrane lipid peroxidation (Purdy et al., 2010). The refrigeration curve used in the present study was approximately 0.13 ◦ C/min until temperature stabilization at 5 ◦ C, which was achieved after 4 h of processing. However, the Botutainer® system provides a decrease in cooling rate starting from 15 ◦ C to 5 ◦ C, generating curves of approximately 0.05–0.1 ◦ C/min. As the critical temperature interval for sperm of different animal species ranges from 15 ◦ C to 5 ◦ C, due to plasma membrane phospholipid rearrangement and the possibility of cold shock (Watson, 2000), slower refrigeration curves have been associated with better preservation of bull semen viability and fertility (Januskauskas et al., 1999). Therefore, the adequacy of the refrigeration curve explains the similar MOT and RAP values observed during the first 6 h of storage regardless of the extender in use.

The differences observed in the PM, VCL, ALH and LIN variables among the different extenders at each experimental time point were similar to those observed in previous studies evaluating the same extenders for semen cryopreservation. Crespilho et al. (2012b) noted more progressive and linear motion along with lesser rates of post-thawing ALH and VCL for cryopreserved bull sperm stored in Botu-Bov® compared to sperm processed in Trisegg yolk-fructose extender. In the previous study it was concluded that the lipid particles found on egg yolk based extenders could also have a deleterious role on sperm motility, acting as a physical barrier for spermatozoa. For this reason, the lesser viscosity of the Botu-Bov® medium (clarified by centrifugation) allowed greater preservation of post-thaw sperm movement. Greater post-thawing MOT, PM, VCL, ALH and LIN values were also associated with the use of Botu-Bov® containing 20% egg yolk compared to Bioxcell® medium composed of soy lecithin (Celeghini et al., 2008). Thus, it has been verified that BB confers greater sperm protection than does TRIS medium when used for refrigeration, as was previously observed for bull semen cryopreservation. Samples refrigerated in BB-L exhibited intermediate motion variables, with lesser PM rates at 24 and 48 h compared to TRIS but greater preservation of sperm linearity (P < 0.05) at all the experimental time points. The kinetic quality promoted by the BB-L extender is probably related to the properties of its specific components, which are the same as those in Botu-Bov® except for the substitution of egg yolk for soy lecithin. Even though significant decreases in MOT, RAP and PAMI were observed over the course of processing regardless of the extender in use in the present study, these variables were less at 6 h when the BB-L medium was used than when other treatments were used. Soy lecithin has been used as a phospholipid source in freezing extenders for bull (Amirat et al., 2005; Crespilho et al., 2012b), ram (Sharafi et al., 2009) and stallion (Papa et al., 2010) semen as a substitute for compounds of animal origin. However, apart from inconsistent results from different studies regarding the effectiveness of lecithin as a substitute for egg yolk in cryopreservation extenders (Crespilho et al., 2012b; Leite et al., 2010), no other studies have focused on the use of this plant compound for the

Please cite this article in press as: Crespilho, A.M., et al., Sperm fertility and viability following 48 h of refrigeration: Evaluation of different extenders for the preservation of bull semen in liquid state. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.020

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Table 2 Conception rate and probability of pregnancy following synchronization of the stage of estrous cycle and time of ovulation when using different methodologies and extenders for bull semen preservation. SemenExtender

Conceptionn/n (%)

Probability of gestation (95% confidence interval)

P-value

TRIS-C TRIS-R Botu-Bov® BB-L

101/253 (39.92)a 59/233 (25.32)b 65/247 (26.32)b 80/240 (33.33)ab

Reference 0.479 (0.318–0.721) 0.518 (0.347–0.772) 0.748 (0.506–1.106)

– 0.0004 0.0013 0.1457

Different letters (a,b ) in the same column indicate statistical differences. TRIS-C = Tris-fructose-egg yolk used for sperm cryopreservation; TRIS-R = Tris-fructose-egg yolk used for semen refrigeration; BB = Botu-Bov® ; BB-L = BotuBov® -Soy Lecithin.

maintenance of refrigerated bull semen and the interaction of this methodology on pregnancy rates in FTAI programs in beef cattle. Paz et al. (2010) tested different lecithin sources and concentrations for ram semen preservation at 5 ◦ C for 6 h (based on the parameters of total motility and plasma membrane integrity) and obtained similar results when comparing a 2% lecithin-based extender to a control group containing egg yolk. Conversely, Paulenz et al. (2002) observed greater preservation of motility and PAMI in ram sperm maintained in a liquid state for time periods greater than 30 h when using Tris-egg yolk medium compared to media based on milk or sodium citrate, regardless of storage temperature. Superior results have also been associated with the use of egg yolk, not only for storage of bull sperm as a liquid but also for cryopreservation (Blackshaw et al., 1957; Foote, 1970). Previous study comparing the same extenders with bull semen cryopreservation observed only 20% of semen samples cryopreserved in egg yolk based extenders did not reach the minimum of 50% total motility post-thaw, while 80% of sperm batches cryopreserved using the extender with soy lecithin as a lipoprotein source had sperm quality below to the international standards required for AI programs (Crespilho et al., 2012b). It has been speculated that these superior MOT, PM and PAMI values in post-thawed bull semen are because the lipoproteins present in egg yolk have greater quality than those contained in soy lecithin (Leite et al., 2010). This has led to the proposal that this superiority might also be extrapolated to media used for bull semen refrigeration. The accumulation of toxic compounds associated with cell death and products generated by sperm metabolism (including ROS) are the main factors that cause the inevitable decrease in sperm viability during the refrigeration period (Nair et al., 2006). These compounds produce an irreversible decrease in sperm motility in cooled bull semen (Almquist and Wickersham, 1962; Foote, 1970). Oxidative stress was indirectly evaluated in the present study through the measurement of malondialdehyde (MDA), one of the major products of sperm membrane lipid peroxidation (Nichi et al., 2007). Regardless of the source (polyunsaturated fatty acids from sperm cell membranes, from extenders or both), lipid peroxidation induced by ROS not only disrupts sperm motility, but also impairs all the sperm functions which are dependent on the integrity of plasma membrane, including sperm-oocyte

fusion and ability to undergo acrosomal exocytose (Bansal and Bilaspuri, 2011). In the present study, sperm maintenance in a soy lecithin-based extender was associated with lesser relative (the mean concentration for each treatment at each of the three experimental time points) and absolute (throughout the 48-h refrigeration period; P = 0.8907) lipid peroxidation rates. However, a significant increase (P < 0.05) in MDA production was associated with the maintenance of refrigerated bull semen in egg yolk-based extenders, with results similar to those previously reported for refrigerated bull and antelope semen (Nair et al., 2006). Chatterjee and Gagnon (2001) observed a 2.4-fold increase in the production of O2− ions during bovine semen refrigeration, which likely underlies the increase in total ROS production during refrigerated storage and emphasizes the need for an adequate refrigeration extender to ensure efficient sperm preservation. Kadirvel et al. (2009) observed that the generation of ROS and sperm membrane lipid peroxidation both undergo a significant and linear increase between 0 and 72 h of the refrigeration process for antelope semen and that both of these events are negatively correlated with sperm motility, mitochondrial membrane potential and sperm chromatin integrity. Kumaresan et al. (2009) obtained similar results with refrigerated boar semen, confirming the significant increase in MDA concentrations during the refrigeration period. ROS were also cited as the main cause of the deterioration in the quality of refrigerated dog semen (Michael et al., 2009). Considering that excessive ROS production causes a reduction in sperm quality, protein oxidation, damage to sperm DNA (Morte et al., 2008) and a consequent decrease in bull fertility rates (Waterhouse et al., 2010), it is possible that quantifying ROS and/or lipid peroxidation may be an efficient technique for assessing sperm quality and, therefore, more accurately identifying differences in fertility rates achieved by use of different semen samples. In a previous study, bull semen cooled for only 24 h enhances the pregnancy rate in cows estrous-synchronized for FTAI, representing a highly viable alternative to conventional frozen-thawed bull semen in terms of both economic and biological comparisons (Crespilho et al., 2012a). In the present study, a greater probability of pregnancy (P < 0.05) was achieved with the use of semen samples diluted in BBL compared to TRIS or BB after 48 h of refrigeration. These results are likely due to less production of ROS in the soy

Please cite this article in press as: Crespilho, A.M., et al., Sperm fertility and viability following 48 h of refrigeration: Evaluation of different extenders for the preservation of bull semen in liquid state. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.020

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lecithin-based medium than in the egg yolk-based extenders. Stradaioli et al. (2007) reported greater concentrations of the antioxidant glutathione in bull semen cryopreserved with the lecithin-based extender Bioxcell® than in samples processed using TRIS media, suggesting that semen frozen in egg yolk-based extenders has a lesser capacity for ROS neutralization, which can impair fertility. The increased membrane lipid peroxidation rates may be associated with cold shock that occurs during refrigeration, resulting in the premature capacitation of bull spermatozoa (Chatterjee and Gagnon, 2001); this represents one of the main causes for the decrease in fertility rates with AI. In this context, results suggest that even with greater sperm preservation capacity (as indicated by greater rates of motility and PAMI), egg yolk-based media do not provide efficient protection against the production of ROS, which leads to sperm alterations that directly impact fertility. It has been speculated that the primary means by which ROS reduce the fertility of semen subjected to refrigeration or long-term liquid storage is its impact on sperm DNA integrity (Jackson et al., 2010). In fact, oxidative stress has been implicated as the major source of sperm DNA fragmentation (Sakkas and Alvarez, 2010). Furthermore, it has been previously demonstrated that sperm showing acceptable morphological and functional traits may show fragmented DNA, with significant impact on fertility (Tesarik et al., 2004). Given that C/AI fertility rates using refrigerated bull semen were similar (BB-L) or inferior (BB and TRIS) to those obtained using cryopreserved semen, the initial hypothesis proposed in the present study has been rejected. Extenders used for conventional sperm cryopreservation do not confer the protection necessary to maintain cooled sperm over long periods of time. According to Vishwanath and Shannon (2000), the rapid decrease in the fertility of sperm stored in the liquid state occurs due to extracellular and intracellular oxidative stress, consistent with the results obtained in the present study. 5. Conclusion Although egg yolk-based extenders provided greater preservation of motility and bull sperm integrity during the refrigeration process, a soy lecithin-based medium was the most effective at preventing lipid peroxidation of the sperm membrane, guaranteeing conception rates comparable to those obtained from conventionally cryopreserved semen. The substitution of cryopreserved bull semen samples for 48-h refrigerated semen samples did not increase C/AI rates in the FTAI program, indicating the need for further studies exploring not only new media for the maintenance of refrigerated sperm but also the relationship between the refrigeration period and the fertility of bull semen when used for AI. Acknowledgements The authors would like to thank the São Paulo Research Foundation (FAPESP, São Paulo, Brazil) for financial support (grant number 06/61153-5) and the Braido Ranch and Stud Farm (Cerqueira César, São Paulo, Brazil) and the Estrela

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do Guaporé Ranch (Comodoro, Mato Grosso, Brazil) for the facilities and for providing the animals used in this study.

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Please cite this article in press as: Crespilho, A.M., et al., Sperm fertility and viability following 48 h of refrigeration: Evaluation of different extenders for the preservation of bull semen in liquid state. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.020