This study evaluated different methods of frozen-thawed sperm selection and capacitation in goats for further use in reproductive biotechnologies. In Experiment 1, semen was processed by the following techniques: mini-Percoll, swim-up, or washing by centrifugation. In Experiment 2, mini-Percoll selected-sperm was subjected to capacitation induction by incubation with: 50 g/mL heparin, 10 M, 50 M, or 100 M of sodium nitroprusside (SNP). Motility, vigor, acrosome, and plasma membrane (PM) integrity were evaluated after thawing and after each treatment of sperm selection or capacitation. In Experiment 1, washing by centrifugation presented greater (43%; P < 0.05) spermatozoa recovery rate than the other treatments. The swim-up technique showed the lowest (P < 0.05) progressive motility (41%). Spermatozoa presenting both intact PM (P = 0.0002) and acrosome (P = 0.0004) showed an interaction effect between the buck and swim-up technique. In Experiment 2, the addition of 100 M SNP resulted in greater (P < 0.05) motility and vigor (38%; 4.7), respectively, than did heparin (28%; 4.3). An enhancement (P < 0.05) in vigor was obtained after all treatments in comparison with the evaluation after thawing (3.3). In conclusion, mini-Percoll was better than swim-up for preparing frozen-thawed goat sperm, whereas washing by centrifugation technique presented similar rates to mini-Percoll and could also be used. The use of 100 M SNP resulted in better motility and vigor than heparin treatment.

Background: The success of fertilization is directly associated with semen quality and the sperm preparation. Considering the common use of cryopreserved spermatozoa, there is a need to develop strategies for sperm preparation in order to achieve a sperm sample of high quality through a rigorous selection of sperm. Thus, sperm cells are being more extensively investigate. This study aimed evaluating the influence of different sperm selection techniques on ram sperm parameters in semen preparation.Materials, Methods & Results: Frozen-thawed commercial semen from 10 Santa Inês rams was subjected to either: swim-up, Percoll, mini-Percoll, sperm washing by centrifugation or a control group. After each technique, samples were incubated at 37°C for 1 h, 2 h and 3 h. At post-selection moment (0 h) and at each interval, sperm recovery rate, motility, capacitation and plasma membrane (PM) integrity were analyzed. The lowest (P < 0.05) recovery rate was recorded after swim-up (1.0 ± 0.3%), whilst the others were similar (P > 0.05). Most part of motility parameters were not affected (P > 0.05) by the technique at 0 h; just swim-up obtained higher (P < 0.05) values for VSL (41.8 ± 11.1) and VAP (46.9 ± 11.2). Overall, swim-up presented higher (P < 0.05) values for most of motility parameters over time of incubation. The control group led to more (P < 0.05) capacitated cells (46.8 ± 2.3%), whilst Percoll, mini-Percoll and swim-up were similar (~33%; P > 0.05), regardless of interval incubation. However, the latter three techniques presented more (P < 0.05) reacted cells. Swim-up recovered a higher (P < 0.05) number of intact cells (32.1 ± 6.4%), and Percoll, mini-Percoll and control group were similar (P > 0.05).Discussion: The present study evaluated the sperm cell and its preparation for receiving the oocyte under optimal conditions. evaluate the sperm cell and its preparation for receiving the oocyte under optimal conditions. Although the swim-up technique promoted higher rates for some of the sperm parameters evaluated, Percoll protocols are the most widely used procedures for selection during the sperm preparation in many species, possibly because of its greater speed, practical method and convenience compared to the swim-up technique. Percoll and mini-Percoll recovered approximately 10 times more cells than swim-up, which is an important feature to be considered during sperm preparation for in vitro fertilization (IVF), being possible to use only one semen straw. The high capacitated and acrosome reaction rates observed after the treatments in the current study, are probably reinforced by changes in sperm cells caused by the cryopreservation process. In order to strength the evidence that frozen-thawed sperm, even after selection, is sensible and reactive to capacitation-like events, we demonstrated the capacitated and acrosome-reacted cell values immediately after the selection treatments behaved differently than when authors used ram fresh sperm. Possibly, this capacitation-like changes observed in frozen-thawed sperm occurs regardless of the selection treatment used. Analyzing the motility parameters immediately after the selection, all treatments maintained or increased the rates compared to the control group. The swim-up, mini-Percoll and Percoll did not differ in any parameters. Given that Percoll and mini-Percoll did not show differences in relation to swim-up for motility parameters, such techniques can be used to replace the latter, obtaining similar sperm samples with good quality. However, swim-up technique involves a procedure that recovers a clean fraction without debris and other types of cells, with high rate of mobile sperm with excellent quality, reason why it can justify the higher recovery of intact spermatozoa after the technique.

This study evaluated the effect of increasing centrifugal force and reducing centrifugation time and volume in Percoll protocols on ram sperm parameters. Commercial semen of Santa Inês rams were used and five treatments were performed: traditional Percoll and mini-Percoll (MP) techniques (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). At post-thawing (PT) and post-selection protocols (0h), samples were assessed for spermatozoa recovery rate, motility, plasma membrane (PM) integrity, sperm capacitation and morphology and incubated at 37 C for 1, 2 and 3h. The sperm recovery rate averaged 9.1±1.4%, and most motility parameters were similar (P> 0.05) among protocols. VCL (µm/s) was higher (P< 0.05) after MP-II, III and IV (66.1±4.5) than traditional Percoll (46.3±4.9). Capacitation status and PM integrity were similar (P> 0.05) among treatments. For the first time, we have demonstrated the reduction of the gradient volume and centrifugation time associated with an increase on centrifugation force at Percoll can be successfully used for frozen-thawed ram sperm selection. MP may be used instead of traditional Percoll, decreasing costs and semen handling time.

Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modifications that render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium, heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PM and would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes in bull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quickly than buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similar whether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by mini-Percoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status and sperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed sperm after mini-Percoll processing followed by incubation with or without heparin supplementation.Materials, Methods & Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percoll and supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes), capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and 18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P < 0.05) in most of the sperm motility parameters after mini-Percoll. Conversely, ovine samples presented the highest (P < 0.05) rate of acrosome-reacted cells after mini-Percoll. Heparin supplementation did not affect most of the parameters evaluated (P > 0.05). In caprine and bovine species, a lower (P < 0.05) rate of sperm agglutination was observed in the presence of heparin at 18 h of incubation. In the absence of heparin, ovine samples showed a higher (P < 0.05) agglutination rate compared to the bovine species after long incubation period.Discussion: The present study compared sperm parameters (sperm kinematics, agglutination rate and capacitation status) of ruminant frozen-thawed sperm after mini-Percoll selection followed by in vitro incubation with or without heparin supplementation. In this study, it was observed the same rate of capacitated cells after the sperm selection (min-Percoll) between ruminant species. This indicate that the capacitation process occurs similarly between ruminant species, refuting the first hypothesis of this study. The presence of heparin did not influence the capacitation status of ruminant frozen-thawed sperm after mini-Percoll selection, it demonstrates that the second hypothesis was supported by this study making more economic and practical the use of ruminant frozen-thawed semen. The absence of heparin in the incubation medium did not harmed the capacitation status and sperm agglutination of ruminant frozen-thawed sperm. This supported the third hypothesis of the current study and indicate that the use of mini-Percoll technique regardless the presence of heparin could be a useful alternative for the preparation of ruminant frozen-thawed sperm. In conclusion, the capacitation status of ruminant frozen-thawed sperm is similar whether or not heparin is present after the mini-Percoll technique.