Boar sperm motility is modulated by CCK at a low concentration of bicarbonate under capacitation conditions.

In a previous study, our group detected the cholecystokinin (CCK) protein in the porcine oviduct. This fact, together with the involvement of CCK in the regulation of sperm protein tyrosine phosphorylation by the modulation of HCO3 - uptake (in mice and humans) suggests a role for CCK during sperm capacitation. Therefore, on the one hand, the expression of CCK receptors (CCK1R and CCK2R) on boar testes has been investigated and probed; on the other hand, boar spermatozoa (from seminal doses of 1-day and 5-day storage) were exposed to different concentrations of CCK (0-control, 25 or 50 µM) in a medium supporting capacitation supplemented with 0, 5 or 25 mmol/L of HCO3 - for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 µM of CCK) were observed when HCO3 - was absent in the media (p > .05). However, the results showed that when the media was supplemented with 5 mmol/L HCO3 - in 1-day seminal dose storage, the linearity index (LIN, %), straightness index (STR, %) and oscillation index (WOB, %) (sperm kinetics parameters) increased in the presence of CCK regardless the concentration (p < .05). Nevertheless, CCK in sperm from 5-day storage only increased the WOB parameter in comparison to the control (p < .05). Furthermore, the average amplitude of the lateral displacement of the sperm head (ALH, µm) and curvilinear velocity (VCL, µm/s) decreased when CCK was present, depending on its concentration and sperm aging (1-day vs. 5-days) (p < .05). In the case of the media supporting capacitation supplemented with 25 mmol/L HCO3 - , any differences were observed except for sperm viability in the 5-day seminal doses, which increased in the 50 µM-CCK group compared to the control (p < .05). In conclusion, these data suggest an implication of CCK protein during sperm capacitation under low bicarbonate concentration increasing the sperm linear trajectory.

Effect of boar seminal dose type (cervical compared with post-cervical insemination) on cooling curve, sperm quality and storage time.

Seminal doses used for cervical and post-cervical artificial insemination (CAI and PCAI, respectively) vary in volume, the number of spermatozoa and packaging. The aim was to evaluate the outcomes when there was use of routine processing procedures for CAI- and PCAI-doses. Two different types of seminal doses were processed: 1) CAI: 2.7 × 109 sperm/80 ml; 2) PCAI: 1.5 × 109 sperm/45 ml. In Experiment 1, the cooling curve of seminal doses during processing occurred in two phases: 1st) At room temperature (23.4 ±â€¯0.5 °C) from 0 (just after packaging) to 120 min; 2nd) At refrigeration (15.7 ±â€¯0.8 °C) from 121-240 min. For the PCAI-doses, the time required to reach room temperature was 47 min compared to 107 min for CAI-doses (decreasing velocity of 0.093 °C/min and 0.048 °C/min, respectively). During refrigeration, for the PCAI-doses the time required to reach the desired preservation temperature was 20 min less than for CAI-doses (PCAI: 90 min, 0.074 °C/min; CAI: 110 min, 0.066 °C/min). In Experiment 2, sperm motility, kinetic parameters and acrosome damage for both types of doses were evaluated at 0, 24, 48 and 72 h of refrigeration. Also, morphology, pH, and osmolality were assessed at 0 and 72 h. Values for all these did not differ between CAI- and PCAI-doses. In conclusion, PCAI-doses took less time than CAI-doses to reach the desired temperature, but sperm quality was similar for CAI- and PCAI-doses during storage. Nevertheless, the different cooling curves should be taken into consideration for further investigation.

Effect of temperature and cell concentration on dolphin (Tursiops truncatus) spermatozoa quality evaluated at different days of refrigeration.

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Sperm cryopreservation in the bottlenose dolphin (Tursiops truncatus) has been developed for the exchange of gametes within the ex situ population. The aim of this study was to develop an effective method for refrigeration of bottlenose dolphin spermatozoa diluted in a commercial extender (BTS). In Experiment 1, the effect of temperature (5 compared with 15 °C) on sperm quality was evaluated during 7 days of storage at 100 × 106 spermatozoa/ml. In Experiment 2, the effect of the storage concentration (100 × 106 compared with 20 × 106 spermatozoa/ml) on sperm quality was assessed during 7 days of storage at 5 °C. In Experiment 1, total motility (including % of rapid sperm) was greater at 5 than 15 °C. When the effect of storage concentration was evaluated (Experiment 2), total motility and ALH were greater at the higher storage concentration (100 × 106 spermatozoa/ml). For both experiments, values for viability, acrosome integrity, and normal morphology variables were consistent throughout the 7 days of refrigeration. In Experiment 3, a microbiological study was performed to evaluate the effect of the refrigeration temperature and days of storage on bacterial growth. The results of microbiological analysis indicated there was Staphylococcus aureus in some samples, however, there was no effect of temperature or days of refrigeration. In conclusion, bottlenose dolphin semen can be refrigerated for a short to medium period of storage and there is maintenance of functionality of sperm when stored at 100 × 106 spermatozoa/ml at 5 °C.

Seminal plasma mitigates the adverse effect of uterine fluid on boar spermatozoa.

After natural or artificial insemination, spermatozoa start their journey within the uterus to reach the site of fertilization, but only few of them attain this goal. Part of this spermatozoa loss happens in the uterus, in which uterine fluid (UF) seems to be involved. It is known from other species that UF provokes damage to spermatozoa, which is avoided when seminal plasma (SP) is present. Therefore, the aim of the present study was to evaluate the effect of UF on the quality of ejaculated (previously contacted with SP) and epididymal (without previous contact with SP) boar spermatozoa analyzing motility, kinetic parameters, viability and acrosome integrity in the presence or absence of SP over time. Three experimental groups were evaluated in each source of spermatozoa (ejaculated and epididymal): 1) Control: spermatozoa with 20% of SP; 2) UF: spermatozoa with 20% of UF; and 3) UF-SP: spermatozoa with 20% of SP and 20% of UF. Total and progressive motility, kinetic parameters (VCL, VSL, VAP, LIN, STR, WOB, and BCF), viability and acrosome damage were analyzed at 15, 60, 120 and 180 min after incubation. Total and progressive motility decreased when ejaculated spermatozoa were incubated in UF in contrast to control and UF-SP groups (p < 0.0007), with no differences between control and UF-SP. The VCL decreased in the UF group compared to the control and UF-SP groups in ejaculated spermatozoa (p = 0.0002). The VSL, VAP, LIN and STR kinetic parameters were greater when ejaculated spermatozoa were incubated in the UF-SP group than in the UF group (all: p ≤ 0.02). Acrosome damage increased in ejaculated and epididymal spermatozoa incubated in the UF group compared to the control and UF-SP groups (both: p < 0.0001). Also, the viability of epididymal spermatozoa decreased in the UF group, while it did not change in the control and UF-SP groups (p = 0.0004). The rest of the parameters in either ejaculated or epididymal spermatozoa did not differ between experimental groups, except for WOB when epididymal spermatozoa were used (UF-SP higher than the control group, with UF being similar for both; p = 0.03). In conclusion, both ejaculated and epididymal spermatozoa are affected by UF, suggesting a negative effect on their quality. This negative effect is reduced by the presence of SP, improving the spermatozoa functionality, preserving motility, viability and acrosome integrity.

Sedation or anaesthesia decrease the stress response to electroejaculation and improve the quality of the collected semen in goat bucks.

Electroejaculation (EE) is stressful and probably painful; thus the administration of anaesthesia is recommended to decrease those negative effects. However, anaesthesia has a direct risk of provoking death, but sedation is less risky than anaesthesia. At the same time, α2-adrenergic agonists may improve semen quality. Therefore, the aim of the present study was to compare the physiological and behavioural responses indicative of stress and possibly pain, and the semen quality in electroejaculated untreated, anaesthetised or sedated goat bucks. Semen was collected from eight bucks using three different procedures in all them (EE in untreated bucks, EE under sedation or EE under general anaesthesia). The number of vocalizations during EE and the behavioural pattern before and after procedures were recorded. Pain visual analogue scale (VAS) score was also determined during EE. Rectal temperature, heart rate, serum cortisol concentration, biochemical and haematological parameters were measured before and after each procedure, and sperm characteristics were determined. Bucks vocalised more often when untreated than sedated or anaesthetised (P<0.02). The pain VAS score was greater when bucks were untreated than sedated or anaesthetised (P<0.002). The rectal temperature, heart rate, total protein, albumin and haemoglobin concentrations were greater when bucks were untreated than anaesthetised or sedated (P<0.02). Serum cortisol increased after EE (P=0.0006), without differences between procedures. The frequency and duration of lying down after EE were greater when bucks were anaesthetised than sedated or untreated (P<0.05), and were also greater when bucks were sedated than untreated (P<0.05). The number of times that the animal tried to stand up after EE was greater when bucks were anaesthetised than sedated or untreated (P<0.02). The sperm mass motility was greater when bucks were anaesthetised or sedated than when they were untreated (P=0.048). When animals were sedated, the ejaculate contained more spermatozoa with functional plasma membrane (P=0.03) and morphologically normal (P=0.05) than when they were untreated. In conclusion, general anaesthesia and sedation decreased the stress and probably the pain response provoked by EE and especially sedation improved the quality of the semen collected.

Ejaculation does not contribute to the stress response to electroejaculation in sheep.

Electroejaculation procedures (EEPs) provoke stress; nevertheless, ejaculation produces physiological changes similar as those usually used to measure stress responses. The application of EEP to animals that cannot ejaculate-as ewes-may be useful to discriminate the responses induced by ejaculation from those provoked by EEP. The aim was to determine the stress response to EEP in rams and ewes. The EEPs were applied to 10 rams and 10 ewes during the non-breeding season, and the number of vocalizations, the heart rate, rectal temperature, serum cortisol concentration, biochemical and haematological parameters were measured. Overall, EEP provoked increases in cortisol concentration, glycaemia, rectal temperature and concentration of creatine kinase (all them: p < .0001) as well as relative concentration of granulocytes (p = .003) and absolute granulocyte concentration (p = .0002) in both, rams and ewes. Heart rate, relative concentration of lymphocytes (p = .001), haematocrit (p = .02) and haemoglobin (p = .045) decreased in animals from both genders after EEP. Besides, cortisol (p < .0001), rectal temperature (p = .002) and glycaemia (p = .001) were greater in ewes than rams, and creatine kinase also tended to be greater in ewes than rams (p = .054). On the other hand, the number of animals that vocalized (p = .006), white blood cells (p = .02) and absolute lymphocytes (p = .02) were greater in rams than ewes. The general trends show a similar pattern of stress responses in animals from both genders. Therefore, we concluded that ejaculation does not contribute to the stress response provoked by the EEP. This procedure also provokes muscular damage and probably pain.