K. L. Goularte

Methods of cryopreservation of Tambaqui semen, Colossoma macropomum

This study compared three different techniques for sperm cryopreservation of Tambaqui (Colossoma macropomum). Semen was diluted in Beltsville Thawing Solution with the addition of dimethyl sulfoxide (DMSO) at various concentrations (5%, 10%, 15% and 20%). Cryopreservation was performed using three methods: Box Conditioner Method with straws at a 5 cm distance from liquid nitrogen vapor (N2L); Dry Shipper Method placing the straws inside the machine; Vitrification Method placing the straws directly into N2L, amounting to 12 treatments (four DMSO concentrations×three freezing methods). The samples were evaluated for analysis of sperm quality in vivo and in vitro. Use of the Vitrification Method at different concentrations of DMSO provided the least values in the different evaluations. Fertilization, hatching rates and plasma membrane integrity using the Box Conditioner Method with 5% and 10% DMSO did not differ (P>0.05) but use of the concentration of 5% DMSO resulted in greater values than the other treatments (P<0.05) as well as for sperm motility and latency time (P<0.05), although sperm viability was superior using the Dry Shipper Method with 20% of the cryoprotectant. Mitochondrial functionality was impaired by use of the Vitrification Method with all DMSO concentration tested showing the most desirable values when the Box Conditioner Method was used with 5%, 10%, 15% DMSO and the Dry Shipper Method was used with 10% and 15% DMSO. Considering the variables evaluated, the use of the Box Conditioner Method is associated with enhanced Tambaqui semen quality with freeze concentrations of 5% and 10% DMSO.

Association between the presence of protein bands in ram seminal plasma and sperm tolerance to freezing.

This study evaluated associations between the presence of protein bands in ram seminal plasma and the quality of sperm frozen with distinct extenders. Ejaculates were frozen in a Tris-egg yolk based extender, including either 5% glycerol or 100mM trehalose. Seminal plasma samples were submitted to unidimensional electrophoresis. Pre-freezing and post-thawing sperm quality was similar between extenders (P>0.05). A total of 26 bands were identified in ram seminal plasma. Pre-freezing sperm motility was increased when the 15, 19 and 80kDa bands were present in seminal plasma (P<0.05). The presence of an 11kDa band in seminal plasma was associated with reduced pre-freezing membrane integrity (P<0.05). After thawing, both sperm motility and membrane integrity were reduced when a 24kDa band was present in seminal plasma (P<0.05). Post-thawing acrosome integrity was greater in the presence of a 31kDa band in seminal plasma (P<0.05). Regardless of the cryoprotectant included in the freezing extender, these six bands may be potential markers for ram sperm tolerance to freezing.

Effect of egg yolk plasma on dog sperm cryopreservation

This study evaluated the quality of frozen‐thawed dog spermatozoon after the inclusion of egg yolk plasma (EYP) instead of whole egg yolk (EY) in the cryopreservation extender and after distinct periods of exposure to EYP. Seven mongrel dogs were used as sperm donors, and EYP was obtained by centrifugation. In Experiment 1, post‐thawing sperm motility (MOT) and integrity of membrane (INT) and acrosome (ACR) were superior for spermatozoon extended with 20% EYP T2 than with 20% EY (P < 0.05), although normal sperm morphology (MOR) did not differ (P > 0.05). In Experiment 2, after ejaculates extended with 20% EYP were cooled at 5°C for 2, 6 and 10 h before freezing, MOT, INT and ACR were similar among periods (P > 0.05). Thus, dog spermatozoon extended with 20% EYP can be kept cooled for up to 10 h prior to freezing, achieving post‐thawing quality greater than that obtained with the inclusion of EY in freezing extenders.

Boar sperm quality after supplementation of diets with omega‐3 polyunsaturated fatty acids extracted from microalgae

This study evaluated effects of diet supplementation with omega‐3 polyunsaturated fatty acids (PUFA) from microalgae on boar sperm quality. Two groups of boars (n = 3 each) were fed during 75 days either a commercial diet (control), or the same diet supplemented with omega‐3 PUFA from the heterotrophic microalgae Schizochytrium sp. (120 g/kg). Sixteen ejaculates were collected per boar. Some sperm kinetics parameters were inferior for supplemented than for control boars (p < .05): distance average path; distance in both curved and straight line; velocity average path, velocity in both curved and straight line; and amplitude of lateral head displacement. Spermatozoa from supplemented boars presented lower mitochondrial functionality, but greater membrane fluidity compared to the control group (p < .01). Membrane and acrosome integrity, production of reactive oxygen species and lipid peroxidation did not differ (p > .05). Serum cholesterol levels were greater (p < .05) for supplemented than for control boars at the 30th and 60th d of supplementation, but levels of triglycerides and IGF‐1 did not differ (p > .05). Compared to the control, spermatozoa of supplemented boars were slower, travelled shorter distances and presented impaired energy metabolism, but their greater membrane fluidity may potentially favour their cryopreservation.

The Use of Antibiotics in Cryopreservation of Ram Sperm

Antibiotics are used in extenders for sperm cryopreservation, however no specific regulation has been established for ram sperm. This study evaluates the effects of antibiotics in ram sperm extenders for bacterial control and its effect on sperm viability. Sperm from five rams was collected, cooled and frozen using extenders with the following antibiotics: 100,000 IU/mL penicillin and 100 µg/mL streptomycin (PES); 500 µg/mL gentamycin, 100 µg/mL tylosin, 300 µg/mL lincomycin and 600 µg/mL spectinomycin (GTLS); 50 µg/mL ceftiofur sodium (CEF); and 1,000 µg/mL enrofloxacin (ENR). Bacillus sp., Corynebacterium sp., Klebsiella sp. and Staphylococcus sp. were isolated from fresh sperm and perpetual area. For cooled sperm, the antibiotic PES, GTLS and ENR were able to reduce the number of colony forming units per mL (CFU/mL), however, CEF was inefficient in all tested concentrations. Motility of cooled sperm was reduced when GTLS and ENR were used (P 0.05), however, motility was reduced with ENR treatment at doses greater than 50% (P < 0.05). The antibiogram revealed resistance of Staphylococcus sp . and Klebsiella sp. to the penicillin/streptomycin association and to tylosin. Both PES and GTLS were able to control bacterial growth on cooled sperm. However, attention should be given to the antibiotics added to the extenders, which may impair sperm motility.

Motilidade de espermatozoides bovinos após incubação em fluído folicular

The objective of this work was to evaluate sperm cell motility after intrafollicular artificial insemination (IFAI) in vivo or after incubation in follicular fluid in vitro. In the in vivo experiment, IFAI was performed, followed by the recovery of follicular content 1 to 4 hours later, in order to assess sperm motility. In the in vitro experiment, spermatozoa from a pool of commercial frozen-thawed semen were evaluated for their kinetics after incubation for 1 or 3 hours, either pure (pool, control group) or in follicular fluid (FF). A low motility of sperm cells was observed in the FF samples, both in vitro and in vivo. In vitro, the main parameters negatively affected in the sperm cells incubated in FF, compared with the control, were: total motility (TM), progressive motility (PM), curvilinear distance, and straightness, after 1 hour of incubation; and TM, PM, average path velocity, and curvilinear velocity after 3 hours of incubation. The ovarian follicle and follicular fluid do not provide a suitable environment to maintain bovine sperm cell motility.

Expression of paraoxonase types 1, 2 and 3 in reproductive tissues and activity of paraoxonase type 1 in the serum and seminal plasma of bulls

The paraoxonases types 1, 2 and 3 (PON1, PON2 and PON3, respectively) are enzymes that degrade lipid peroxides, preventing oxidative damages relevant for male reproductive function. This study determined the expression of those three paraoxonases in reproductive tissues of bulls and evaluated correlations among the activity of PON1 in the serum and seminal plasma with breeding soundness parameters in bulls. The expression of PON1, PON2 and PON3 was characterised by RT‐PCR in samples of testicular parenchyma, vesicular glands and epididymis collected from three slaughtered bulls. All three paraoxonases were expressed in the testicular parenchyma, PON2 and PON3 were both expressed in the epididymis head and PON3 was also expressed in the epididymis tail. The PON1 activity was determined in samples of serum and seminal plasma from 110 bulls submitted to breeding soundness evaluation. There was a strong correlation (r = .90) between the activity of the PON1 in both serum and seminal plasma (p < .0001). The PON1 activity in the seminal plasma was positively correlated with ejaculates colour, sperm mass activity (p = .04), motility, vigour and viability (all p < .01). Thus, PON1 may be a potential marker for sperm motility and viability in bulls.