Gabriela Liberalino Lima

Dimethylformamide is no better than glycerol for cryopreservation of canine semen.

The objective of this study was to compare the effects of dimethylformamide (DMF) and glycerol in canine (Canis lupus familiaris) semen cryopreservation based on postthaw motility and velocity evaluated by computer-assisted sperm analysis (CASA) and the effects on subjective progressive motility, percentage of live sperm, and plasma membrane functional integrity. The semen was diluted in two steps with an egg-yolk Tris extender containing 6% glycerol or DMF, frozen in 0.25-mL straws, and stored in liquid nitrogen. Immediately after thawing, samples were accessed for subjective sperm motility, sperm membrane functional integrity, percentage of live sperm, and evaluation by CASA. There were differences (P<0.05) between glycerol and DMF with regard to subjective progressive motility (43.1% vs. 21.5%), objective progressive motility (11.8% vs. 6.2%), velocity average pathway (31.1 vs. 23.1 microm/sec), and amplitude of lateral head (3.3 vs. 3.9 microm), which confirmed the efficiency of glycerol. In conclusion, objective analysis performed by CASA confirmed that no benefits were derived by using DMF to replace glycerol for cryopreservation of canine semen.

Effect of centrifugation and sugar supplementation on the semen cryopreservation of captive collared peccaries (Tayassu tajacu)

The present study is aimed at evaluating the effect of centrifugation for seminal plasma removal and the supplementation of fructose or glucose to the Tris-based extender on the kinematic patterns of the motility parameters of frozen-thawed semen obtained from captive collared peccaries (Tayassu tajacu). Semen samples (n = 14) were collected from 10 sexually mature male collared peccaries by electroejaculation. These samples were further evaluated for parameters such as motility, vigor, sperm viability, membrane integrity, and sperm morphology. The samples were divided into four aliquots, and only two of these aliquots were centrifuged. The semen aliquots (centrifuged and raw semen samples) were diluted in Tris-based extenders supplemented with fructose or glucose. Egg yolk (20%) and glycerol (3%) were added to all the samples which were cryopreserved in liquid nitrogen and thawed at 37 °C/1 min. The frozen-thawed semen was evaluated for the same parameters described for the fresh semen. On the other hand, the kinematic motility patterns were evaluated by a computer-aided system. After thawing, it was observed that the values for the total sperm motility were around 30% for all the samples. A negative effect of centrifugation was verified for parameters such as sperm morphology, linearity, straightness, and beat cross frequency (P < 0.05). However, no differences between fructose and glucose were verified for any semen end point (P > 0.05). In conclusion, it is not recommended to centrifuge the ejaculates from collared peccaries prior to conducting the cryopreservative procedures using a Tris-based extender supplemented with fructose or glucose.

Cryopreservation of collared peccaries (Tayassu tajacu) semen using a powdered coconut water (ACP-116c) based extender plus various concentrations of egg yolk and glycerol.

The objective was to determine the effectiveness of a powdered coconut water-based extender (ACP-116c), plus various concentrations of egg-yolk and glycerol, as an alternative for cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were apportioned into aliquots that were diluted in Tris plus 10% egg yolk and 3% glycerol, or in ACP-116c plus 10 or 20% egg yolk and 1.5 or 3% glycerol. Samples were frozen in liquid nitrogen and, after 1 mo, thawed at 37 °C for 1 min. After thawing, samples were evaluated as reported for fresh semen, and also for sperm membrane integrity (fluorescent probes) and kinematic parameters (computerized analysis). Results were presented as means ± SEM. Freezing and thawing decreased sperm characteristics relative to fresh semen. Overall, ACP-116c plus 20% egg yolk and 3% glycerol provided better (P < 0.05) sperm motility and kinetic rating (48 ± 6.1% and 2.8 ± 0.2, respectively) after thawing than Tris extender (30.4 ± 5.7% and 2.4 ± 0.2). However, there were no differences (P > 0.05) among treatments with regard to the other sperm characteristics. Based on computerized motion analysis, total (26.5 ± 5.9%) and progressive (8.1 ± 2.2%) motility were best preserved (P < 0.05) with the above-mentioned treatment. In conclusion, a coconut water-based extender, ACP-116c, plus 20% egg yolk and 3% glycerol, was effective for cryopreservation of semen from collared peccaries.

Objective assessment of the cryoprotective effects of dimethylformamide for freezing goat semen

The aim of this work was to assess the cryoprotective effects of dimethylformamide (DMF) for freezing goat semen, using an objective analysis by computer-assisted sperm analysis (CASA). Twenty-one ejaculates (seven per animal) were collected from three stud bucks with the aid of an artificial vagina and immediately evaluated for gross and microscopic characteristics. The semen was diluted in two steps with a Tris-egg yolk extender containing 6% glycerol or 6% DMF, frozen in 0.50-mL straws, and stored in liquid nitrogen. Samples were accessed for sperm morphology, sperm membrane structural and functional integrity, and by CASA, immediately after thawing. There were differences (P<0.05) between glycerol and DMF with regard to subjective progressive motility (23.9±2.2% vs. 16.6±2.0%), objective progressive motility (3.5±0.4% vs. 1.8±0.3%), linearity (53.9±1.6% vs. 48.1±1.4%) and amplitude of lateral head (2.3±0.1 vs. 2.9±0.1 mm), which confirmed the efficiency of glycerol. In conclusion, dimethylformamide could be used as an alternative cryoprotectant for goat semen freezing. However it was showed that no benefits were derived by using dimethylformamide to replace glycerol at an equal 6% concentration.

Short-term storage of canine preantral ovarian follicles using a powdered coconut water (ACP®)-based medium

The objective was to investigate the use of powdered coconut water (ACP)-based medium for short-term preservation of canine preantral follicles. Pairs of ovaries from mongrel bitches (n=9) were divided into fragments. One ovarian fragment, treated as a fresh control, was immediately fixed for histological analysis, whereas the other six ovarian fragments were stored either in phosphate-buffered saline (PBS; control group) or ACP medium in isothermal Styrofoam boxes containing biological ice packs. The boxes were sealed and opened only after 12, 24, or 36h. After opening each box, the ovarian fragments were submitted to histological analysis. In total, 12,302 preantral follicles were evaluated, with 64.5% primordial, 33.3% primary, and 2.3% secondary follicles. There were multiple oocytes in 1.3% of the follicles analyzed. At 24h, ACP was more efficient in preserving follicular morphology than PBS (P<0.05). Compared with the fresh control group, a significant reduction in the percentage of morphologically normal ovarian follicles was observed for PBS, starting at 24h; however, the decline started only at 36h for the ACP medium. During the experiment, the temperature inside the isothermal boxes increased from 3 to 9 degrees C (P<0.05), despite a constant room temperature. In conclusion, powdered coconut water (ACP) was an appropriate medium for short-term storage of canine preantral ovarian follicles.

Sperm morphological and morphometric evaluation in captive collared peccaries (Pecari tajacu)

ABSTRACT.- Sousa P.C., Santos E.A.A., Souza A.L.P., Lima G.L., Barros F.F.P.C., Oliveira M.F. & Silva A.R. 2013. Sperm morphological and morphometric evaluation in captive collared peccaries ( Pecari tajacu ). Pesquisa Veterinaria Brasileira 33(7):924-930. Laboratorio de Con -servacao de Germoplasma Animal, Universidade Federal Rural do Semi-Arido, BR 110 Km 47, Presidente Costa e Silva, Mossoro, RN 59625-900, Brazil. E-mail: legio2000@yahoo.comThe aim of this study was to compare different staining methods for the evaluation of sperm morphology by light microscopy and also to describe the morphometry of the entire sperm in collared peccaries ( Pecari tajacu ). Semen from 10 males was obtained by electro -ejaculation and evaluated for sperm motility, vigor, and concentration. Semen smears were prepared through three different staining methods: Bengal rose, brome-phenol blue, and eosin-nigrosin. Smears were evaluated under light microscopy and sperm morphologic al-terations were determined in percentage. In addition, sperm morphometric analysis was conducted by light microscopy coupled to image analyzer software. The smears stained with Bengal Rose provide the best results for the visualization of the sperm tail, midpiece, and head. The use of eosin-nigrosin stain did not allow an adequate impregnation, and some sperm presented a few contrasts with the background. A higher incidence of bent coiled tails was verified in the use of brome-phenol blue staining (P<0.05). Through mor-phometric evaluation, it was observed that the tail occupies the greatest proportion (89%) of the sperm which presents a discretely elongated head. According to the results, the use of the Bengal Rose stain is recommended for the morphologic evaluation of the collared peccary sperm.

Morphology, morphometry and ultrastructure of captive six-banded armadillo (Euphractus sexcinctus) sperm.

We analyzed the sperm characteristics of captive six-banded armadillos (Euphractus sexcinctus), by the assessment of sperm morphology, morphometry, and ultrastructure. In general, armadillos ejaculates present more than 80% of sperm within the range considered normal for sperm morphology currently accepted for other mammals. Coiled tails (3.9%) and detached heads (2.8%) were the defects most frequently verified. The morphometric analysis revealed that the total length of six-banded armadillo sperm is 77.6±1.2μm, and the length of the tail is 64.7±1.1μm on average. They also present a big head that corresponds to 16.6% of the entire sperm. Through transmission electron microscopy, we identified the presence of electron lucent points into the nucleus and the presence of about 45 mitochondria spirals in the mitochondrial sheath midpiece as a peculiarity of the six-banded armadillo sperm.

Assessment of sperm survival and functional membrane integrity of the six-banded armadillo (Euphractus sexcinctus)

The objective was to evaluate sperm survival in the six-banded armadillo, using a thermoresistance test, and to compare sugar solutions with varying osmolarities to analyze the integrity of the functional sperm plasma membrane in this species. Twelve ejaculates were obtained from four mature males by electroejaculation and evaluated for sperm motility, vigor, live sperm, and morphology. Sperm survival was evaluated during a thermoresistance test at 34 °C (the body temperature of this species). The functional integrity of the plasma membrane was evaluated by means of the hypo-osmotic swelling test (HOST), using solutions of varying osmolarities (0, 50, 100, and 150 mOsm/L). During the thermoresistance test, at each evaluation, there was a reduction (P < 0.05) in mean values for sperm motility, sperm vigor, and percentage of live sperm (no movement was observed at 360 min). Sperm survival varied among individual armadillos (P < 0.05). In two individuals, sperm vigor was significantly enhanced when semen was diluted in Tris extender. The response of armadillo sperm to the HOST varied among individuals (P < 0.05). On average, maximal values (P < 0.05) of reactive sperm (59%) were detected with 50 mOsm/L solution; furthermore, this concentration had the largest significant positive correlation (r = 0.84) to live sperm percentage. In conclusion, six-banded armadillos had significant individual variation with regard to sperm survival in a thermoresistance test at 34 °C; in some individuals, sperm survived until 360 min. The use of a 50 mOsm/L fructose solution was recommended for conducting a HOST in this species.

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves, straw sizes, and thawing rates.

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (-10 °C/min) or a fast (-40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P<0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P>0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P<0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P>0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples tha wed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P<0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.

Determination of semen characteristics and sperm cell ultrastructure of captive coatis (Nasua nasua) collected by electroejaculation

Despite the wide geographical distribution of coati (Nasua nasua) from the south of Canada to the north of Argentina, studies regarding the reproductive characteristics of this species are extremely limited. The objective of this study was to describe the various characteristics of coati semen by morphometric and ultrastructural analysis. Five mature males were anesthetized and electroejaculated for the collection of semen. Semen was immediately evaluated for color, volume, pH, sperm motility, vigor, morphology, acrosomal integrity, percentage of live cells and hypo-osmotic response by light microscopy. Sperm cell morphometry and ultrastructural analyses were also performed. Observations of seminal characteristics determined by electroejaculation in captive coatis represent a valuable baseline dataset for establishing fertility standards and provide background information that may be useful for assisted breeding programmes in members of the Procyonidae family.